Methods for weed control

ABSTRACT

The invention provides methods for weed control with dicamba and related herbicides. It was found that pre-emergent applications of dicamba at or near planting could be made without significant crop damage or yield loss. The techniques can be combined with the herbicide glyphosate to improve the degree of weed control and permit control of herbicide tolerant weeds.

This application claims the priority of U.S. Provisional PatentApplication 60/811,276, filed Jun. 6, 2006, the disclosure of which isincorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates generally to the field of weed management. Morespecifically, the invention relates to methods for using auxin-likeherbicides such as dicamba for controlling weeds.

2. Description of the Related Art

Weeds cost farmers billions of dollars annually in crop losses and theexpense of efforts to keep weeds under control. Weeds also serve ashosts for crop diseases and insect pests. The losses caused by weeds inagricultural production environments include decreases in crop yield,reduced crop quality, increased irrigation costs, increased harvestingcosts, decreased land value, injury to livestock, and crop damage frominsects and diseases harbored by the weeds. The principal means by whichweeds cause these effects are: 1) competing with crop plants for theessentials of growth and development, 2) production of toxic or irritantchemicals that cause human or animal health problem, 3) production ofimmense quantities of seed or vegetative reproductive parts or both thatcontaminate agricultural products and perpetuate the species inagricultural lands, and 4) production on agricultural andnonagricultural lands of vast amounts of vegetation that must bedisposed of. The damage caused can be significant. For example, it isestimated that between 1972 and 1976 corn yields were reduced by about10% due to weeds (Chandler, 1981).

Among weeds that serve as hosts for crop pests, for example, pepperweedand tansymustard (Descurainia sp.) maintain large populations ofdiamondback moths during the late fall, winter, and spring. They arealso hosts to the turnip aphid and green peach aphid. Several weedspecies of the nightshade family (Solanaceae) are hosts to insects thatcommonly attack eggplant, pepper, potato, and tomato. For example,horsenettle (Solanum carolinense L.) is a host of the Colorado potatobeetle, and black nightshade (S. nigrum L.) is a host of the cabbagelooper. Morning-glory is an important host of insects attacking sweetpotato, especially the highly destructive sweet potato weevil. Ragweedserves as a host for Mansonia mosquitoes, an insect vector for the humandiseases encephalitis and rural filariasis.

Some weeds are undesirable in hay, pastures, and rangelands because ofthe mechanical injury that they inflict on livestock. Woody stems,thorns, and stiff seed awns cause injury to the mouth and digestivetract of livestock; and the hairs and fibers of some plants tend to ballup and obstruct the intestines, especially in horses, causing seriousproblems. Ingested by milk cows, some weeds such as ragweeds, wildgarlic (Allium vineale L.), and mustard, among others, impart adistinctly distasteful odor or flavor to milk and butter. Barbed seeddispersal units may become so entangled in the wool of sheep as togreatly diminish its market value. Parasitic plants, such as dodder(Cuscuta sp.), broomrape (Orobanche sp.), and witchweed, rob their hostplants of organic foodstuffs.

Chemical herbicides have provided an effective method of weed controlover the years. Herbicides can generally be applied pre-emergence and/orpost-emergence. Pre-emergence herbicides are applied in a field before acrop emerges from the soil. Such applications are typically applied tothe soil before, at the same time, or soon after planting the crop. Suchapplications may kill weeds that are growing in the field prior to theemergence of the crop, and may also prevent or reduce germination ofweeds that are present in the soil. Post-emergence herbicides aretypically used to kill weeds after a crop has emerged in the field. Suchapplications may kill weeds in the field and prevent or reduce futureweed germination. In either case, the herbicides may be applied to thesurface of the soil, mixed with the soil, over the top of the plant, orapplied by any other method known to those of skill in the art.

One weed control strategy is to apply an herbicide such as dicamba to afield before sowing seeds. However, after applying the herbicide to afield, a farmer has to wait at least several weeks before sowing thefield with crop seeds such that the herbicide has killed most of theweeds and has degraded so as not injure the sown crop. For example,plants are especially sensitive to dicamba and it has been recommendedthat dicamba formulations such as Banvel™ or Sterling™ be applied 30days prior to planting for controlling weeds. A comprehensive list ofweeds that are controlled by dicamba is available (Anonymous, 2007). Theherbicide is particularly useful for control of taller weeds and moredifficult to control weeds such as purslane, sicklepod, morninglory andwild buckwheat. Dicamba can be used to control weeds not susceptible toother herbicides. Following the application of Clarity™, anotherformulation of dicamba, a minimum accumulation of one inch of rainfallor overhead irrigation followed by a 14 day waiting period for the 4 to8 ounce/acre rates or a 28 day waiting period for the 16 ounce/acrerates has been recommend for controlling weeds in a soybean field (seeTable 22 in VanGessel and Majek, 2005). Also, the Clarity® labelrecommends that it be applied at least 15 days prior to sorghumplanting. Similarly, for cotton, a waiting period of 21 days isrecommended after applying Clarity® or Banvel® to the field, beforeplanting the cotton seeds (Craig et al., 2005, Crop Profile for Cotton(Gossypium hirsutum) in Tennessee,www.ipmcenters.org/cropprofiles/docs/tncotton.html) and no pre-emergenceand post-emergence application are recommended. The waiting period isalso dependent on the crop growing environment at any give time, such asthe type of soil (soil having organic activity will degrade dicambafaster), moisture content, rainfall, temperature, as well as type offormulation and rate of application.

The herbicide 2,4-D has been recommended for controlling certain weedsin a soybean field such as mustard spp., plantains, marestail, and 2,4-Dsusceptible annual broadleaf weeds by applying it 7 to 30 days prior toplanting, depending on rate and formulation (ester or amine) (see Table22 in VanGessel and Majek, 2005).

One method that has been successfully used to manage weeds combinesherbicide treatments with crops that are tolerant to the herbicides. Inthis manner, herbicides that would normally injure a crop can be appliedbefore and during growth of the crop without causing damage. Thus, weedsmay be effectively controlled and new weed control options are madeavailable to the grower. In recent years, crops tolerant to severalherbicides have been developed. For example, crops tolerant to2,4-dichlorophenoxyacetic acid (Streber and Willmitzer, 1989),bromoxynil (Stalker et al., 1988), glyphosate (Comai et al., 1985) andphosphinothricin (De Block et al., 1987) have been developed.

Recently, a gene for dicamba monooxygenase (DMO) was isolated fromPseudomonas maltophilia (US Patent Application No: 20030135879) which isinvolved in the conversion of a herbicidal form of the herbicide dicamba(3,6-dichloro-o-anisic acid) to a non-toxic 3,6-dichlorosalicylic acid.The inventors reported the transformation of the DMO gene into tobaccoand Arabidopsis. The transformed plant tissue was selected on kanamycinand regenerated into a plant. However, herbicide tolerance was notdemonstrated or suggested in immature tissues or seedlings or in otherplants. Pre-emergence herbicide tolerance to dicamba was not described.Transgenic soybean plants and other plants tolerant to application ofdicamba are described in Behrens et al. (2007).

Dicamba is one member of a class of herbicides commonly referred to as“auxin-like” herbicides or “synthetic auxins.” These herbicides mimic oract like the natural plant growth regulators called auxins. Auxin-likeherbicides appear to affect cell wall plasticity and nucleic acidmetabolism, which can lead to uncontrolled cell division and growth. Theinjury symptoms caused by auxin-like herbicides include epinasticbending and twisting of stems and petioles, leaf cupping and curling,and abnormal leaf shape and venation.

Dicamba is one of the many auxin-like herbicides that is a low-cost,environmentally-friendly herbicide that has been used as a pre-emergenceherbicide (i.e., 30 days prior to planting) in dicots and as a pre-and/or post-emergence herbicide in corn, sorghum, small grains, pasture,hay, rangeland, sugarcane, asparagus, turf, and grass seed crops toeffectively control annual and perennial broadleaf weeds and severalgrassy weeds (Crop Protection Chemicals Reference, 1995). Unfortunately,dicamba can injure many commercial crops including beans, soybeans,cotton, peas, potatoes, sunflowers, tomatoes, tobacco, and fruit trees,ornamental plants and trees, and other broadleaf plants when it comesinto contact with them. Soybean and cotton are particularly sensitive todicamba. Thus, applications of dicamba must generally occur severalweeks before planting of sensitive crops to ensure that residual dicambais sufficiently cleared from the crop environment before crops emerge.For post-emergent weed control in corn, dicamba is the 5th most widelyused herbicide for broad leaf weeds. However, although the optimal ratefor broad leaf weed control is between 280 to 560 g/h (grams/hectare),the average use rate in corn is 168 g/h as at higher use rates and undercertain environmental conditions, dicamba can injure corn.

As noted above, current manufacturer's guidelines typically require atleast a 30 day delay between the application of dicamba and the plantingof sensitive crops. This inability to apply dicamba close to the timethat crops are planted delays sowing time and shortens the growingseason, thereby increasing the risk of exposing crops to frost in thefall. The delay also means that the farmers have to go through the fieldtwice; once for planting and once for spraying, thereby increasing fueland wear-tear costs to the farmers. Improvements over the state of theart that would eliminate the delay would positively impact the qualityand quantity of the crop which could result and reduce economic lossesto farmers. More effective weed control would also reduce the risk ofweeds developing resistance to existing herbicides.

SUMMARY OF THE INVENTION

In one aspect, the invention provides a method for controlling weedgrowth in a field comprising: a) applying a herbicidally effectiveamount of an auxin-like herbicide to a crop-growing environment; andplanting a transgenic seed of a dicotyledonous plant expressing anucleic acid encoding dicamba monooxygenase in soil of the crop-growingenvironment, wherein the seed germinates within 30 days or less ofapplying the herbicide and wherein the dicamba monooxygenase comprisesat least 70% sequence identity to the polypeptide sequence of SEQ IDNO:2; and c) allowing the seed to germinate into a plant. In certainembodiments, the seed germinates within four weeks, three weeks, twoweeks, or less than one week after treating the growing environment withthe auxin-like herbicide. The treated growing environment may be, forexample, a field in which a crop is planted. A population of seeds of aplant tolerant to the auxin-like herbicide may be planted in the field.Treating the environment can be carried out according to knowntechniques in the art using, for example, commercially availableformulations of auxin-like herbicides such as dicamba. The environmentincludes an area for which control of weeds is desired and in which theseed of a plant tolerant to the auxin-like herbicide can be planted. Aweed can be directly contacted with herbicide in the environment andsoil in the environment can be contacted with the herbicide, preventingor reducing weed growth in the soil. The step of treating theenvironment with a herbicide may be carried out before, after, orconcurrently with the step of planting the soil with the transgenicseed. The transgenic seed may be planted into soil in the environment,for example, within three weeks before or after treatment, includingfrom between about two weeks, one week and 0 weeks before or aftertreatment, further including from between about 1, 2, 3, 4, 5, or 6 daysbefore or after treatment, including concurrently with treatment. In themethod, the seed may germinate, for example, from between about 30 daysand 0 days after treating the environment, including between about 21,18, 16, 14, 12, 10, 8, 6, 5, 4, 3, 2, 1 and about 0 days after treatingthe environment. The method may further comprise applying one or moreadditional treatments of an auxin-like herbicide after the seedgerminates and/or the plant is growing. In certain embodiments, a secondtreatment is carried out at a time selected from the group consisting ofbetween about the 1 to 2 leaf and 3 to 4 leaf stages, before flowering,at flowering, after flowering, and at seed formation. In one embodiment,the second treatment comprises applying dicamba and/or a2,4-dichlorophenoxyacetic compound (2,4-D).

In a method of the invention, the auxin-like herbicide may be selectedfrom the group consisting of a phenoxy carboxylic acid compound, benzoicacid compound, pyridine carboxylic acid compound, quinoline carboxylicacid compound, and benazolinethyl compound. Examples of a phenoxycarboxylic acid compound include 2,4-dichlorophenoxyacetic acid and(4-chloro-2-methylphenoxy)acetic acid. In certain embodiments, aherbicidally effective amount of 2,4-D and/or(4-chloro-2-methylphenoxy)acetic acid used is between about 2 g/ha(grams/hectare) to about 5000 g/ha, including about 50 g/ha to about2500 g/ha, about 60 g/ha to about 2000 g/ha, about 100 g/ha to about2000 g/ha, about 75 g/ha to about 1000 g/ha, about 100 g/ha to about 500g/ha, and from about 100 g/ha to about 280 g/ha. In one embodiment foundto function particularly well with the invention, dicamba is used as theherbicide. In certain embodiments, a herbicidally effective amount ofdicamba used may be from about 2.5 g/ha to about 10,080 g/ha, includingabout 2.5 g/ha to about 5,040 g/ha, about 5 g/ha to about 2,020 g/ha,about 10 g/a to about 820 g/h and about 50 g/ha to about 1,000 g/ha,about 100 g/ha to about 800 g/ha and about 250 g/ha to about 800 g/ha.

In a method of the invention a plant may be used exhibiting tolerance toauxin-like herbicides including dicamba. Such a plant may comprise anucleic acid encoding a dicamba monooxygenase. In one embodiment, theplant is defined as comprising a nucleic acid encoding a dicambamonooxygenase that has at least 70% identity to a polypeptide sequenceof any one or more of SEQ ID NOs:2, 4, 6, 8, 10 or 12, including atleast about 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% and greater sequenceidentity to these sequences. Polypeptide or polynucleotide comparisonsmay be carried out and identity determined as is known in the art, forexample, using MEGAlign (DNAStar, Inc., 1228 S. Park St., Madison, Wis.53715) with default parameters. Such software matches similar sequencesby assigning degrees of similarity or identity.

The methods of the invention may be used in connection with plants thatexhibit susceptibility to auxin-like herbicides such as dicotyledonous(dicot) plants. In certain embodiments, a dicotyledonous plant is usedselected from the group consisting of alfalfa, beans, broccoli, cabbage,carrot, cauliflower, celery, cotton, cucumber, eggplant, lettuce, melon,pea, pepper, pumpkin, radish, rapeseed, spinach, soybean, squash,tomato, and watermelon. In some embodiments, the dicot is soybean,cotton, or canola.

In another aspect, the invention provides a method for controlling aweed in a field comprising: a) planting a transgenic seed in a field,wherein the seed comprises transgenes conferring tolerance to anauxin-like herbicide and a second herbicide; b) growing the seed into aplant; and c) treating the field with an amount of the auxin-likeherbicide and the second herbicide in amounts effective to control weedgrowth. In some embodiments, the second herbicide may be glufosinate (DeBlock et al., 1987), a sulfonylurea (Sathasiivan et al., 1990), animidazolinone (U.S. Pat. No. 5,633,437; U.S. Pat. No. 6,613,963),bromoxynil (Stalker et al., 1988), dalapon or 2,2-Dichloropropionic acid(Buchanan-Wollaston et al., 1989), cyclohexanedione (U.S. Pat. No.6,414,222), a protoporphyrinogen oxidase inhibitor (U.S. Pat. No.5,939,602), norflurazon (Misawa et al., 1993 and Misawa et al., 1994),or isoxaflutole (WO 96/38567) herbicide, among others. The auxin-likeherbicide and the second herbicide may be applied simultaneously orseparately. In a particular embodiment, the second herbicide isglyphosate and the auxin-like herbicide is dicamba. In one embodiment,the plant comprises a nucleic acid that has at least 70% sequenceidentity to a nucleic acid sequence of any one or more of SEQ ID NOs: 1,3, 5, 7, 9, or 11, including at least about 75%, 80%, 85%, 90%, 95%,97%, 98%, 99% and greater sequence identity to these sequence.

In further embodiments, a plant such as the foregoing is defined ascomprising a transgene conferring glyphosate tolerance. Glyphosateresistant 5-enolpyruvylshikimate-3-phosphate synthases (EPSPS) are wellknown in the art and disclosed, for example, in U.S. Pat. No. 5,627,061,U.S. Pat. No. 5,633,435, U.S. Pat. No. 6,040,497, U.S. Pat. No.5,094,945, WO04074443, and WO04009761. Nucleic acids encoding glyphosatedegrading enzymes, for example, glyphosate oxidoreductase (GOX, U.S.Pat. No. 5,463,175), and nucleic acids encoding glyphosate inactivatingenzymes, such as glyphosate-N-acetyl transferase (GAT, U.S. Patentpublication 20030083480; U.S. Patent Publication 20070079393) andglyphosate decarboxylase (WO05003362 and US Patent Application20040177399) are also known. In certain embodiments, the GAT enzymecomprises the sequence of GAT4601 (SEQ ID NO:19), or is encoded by atransgene comprising the nucleic acid sequence of SEQ ID NO:18. In aparticular embodiment, the GAT polypeptide is expressed using the SCP1promoter.

In the method, treating the field may be carried out at a time selectedfrom the group consisting of between about the 1 to 2 leaf and 3 to 4leaf stages, before flowering, at flowering, after flowering, and atseed formation. Treating the field may further be defined as carried outat a time proximate to step a) such that the seed germinates while theauxin-like herbicide remains in the soil in an amount effective tocontrol growth of the weed. In the method, treating the field may becarried out about three weeks, two weeks 1 week or 0 weeks before stepa). The auxin-like herbicide may be selected from the group consistingof a phenoxy carboxylic acid compound, benzoic acid compound, pyridinecarboxylic acid compound, quinoline carboxylic acid compound, andbenazolinethyl compound.

The phenoxy carboxylic acid compound may be selected from the groupconsisting of 2,4-dichlorophenoxyacetic acid, (4-chloro-2-methylphenoxy)acetic acid, and 4-(2,4-dichlorophenoxy)butyric acid (2,4-DB). Theamount of 2,4-dichlorophenoxyacetic compound used may be lower thanabout 280 g/ha. The amount of 4-(2,4-dichlorophenoxy)butyric acid(2,4-DB) used may be lower than about 1120 g/ha. The amount of(4-chloro-2-methylphenoxy)acetic acid compound used may be lower thanabout 280 g/ha. In one embodiment, the auxin like herbicide is dicamba.The amount of dicamba used may be, for example, from about 2.5 g/ha toabout 10,080 g/ha, including about 2.5 g/ha to about 1040 g/ha, about 5g/ha to about 2040 g/ha, about 10 g/a to about 820 g/h, and about 50g/ha to about 1000 g/ha. The amount of glyphosate may be from about 200g/ha to about 1,600 g/h, including from about 200 g/ha to about 1,000g/h, from about 200 g/ha to about 800 g/h, from about 200 g/ha to about400 g/h, and from about 400 g/ha to about 800 g/h.

In yet another aspect, the invention provides a method for controllingweed growth in a crop-growing environment comprising: a) applying aherbicidally effective amount of an auxin-like herbicide to acrop-growing environment; b) planting a transgenic seed of amonocotyledonous plant comprising a nucleic acid encoding a dicambadegrading enzymatic activity, such as dicamba monooxygenase, in soil ofthe crop-growing environment within 21 days of applying the auxin-likeherbicide, wherein the herbicidally effective amount is an amount thatdoes not damage the transgenic seed or a plant that germinates therefrombut will damage a seed or a plant that germinates therefrom of the samegenotype that lacks the nucleic acid and is planted under the sameconditions as the transgenic seed, wherein the nucleic acid is selectedfrom the group consisting of (1) a nucleic acid sequence encoding thepolypeptide of SEQ ID NO:8, (2) a nucleic acid sequence comprising thesequence of SEQ ID NO:7, (3) a nucleic acid sequence that hybridizes toa complement of the nucleic acid sequence of SEQ ID NO:7 underconditions of 5×SSC, 50% formamide and 42° C., (4) a nucleic acidsequence having at least 70% sequence identity to the nucleic acidsequence of SEQ ID NO:7, and (5) a nucleic acid sequence encoding apolypeptide having at least 70% sequence identity to the polypeptidesequence of SEQ ID NO:8; and c) allowing the seed to germinate into aplant. The nucleic acid sequence having at least 70% sequence identityto the nucleic acid sequence of SEQ ID NO:7 may encode a polypeptidecomprising a cysteine residue at position 112. This embodiment maycombined with any of the methods and compositions provided above.

In particular embodiments of the invention, herbicide treatments tomonocot plants may be made at higher rates and/or in closer proximity toemergence of crops than previously could be made without damaging crops.In specific embodiments, a herbicidally effective amount of 2,4-D and/orMCPA, such as, for example, at least about 200, 300, 300, 500, 590, 650,700, 800 or more g/ha of either or both herbicides, including from about300 to about 1200 g/ha, from about 500 to about 1200 g/ha, from about600 to about 1200 g/ha, from about 590 to about 1400 g/ha, and fromabout 700 to about 1100 g/ha of either or both herbicides. The herbicidemay also be dicamba and the herbicidally effective amount may be, forexample, at least about 168, 175, 190, 200, 225, 250, 280, 300, 400,500, 560 or more g/ha of dicamba, including from about 200 g/ha to about600 g/ha, from about 250 g/ha to about 600 g/ha, from about 250 g/ha toabout 800 g/ha, from about 225 g/ha to about 1120 g/ha, and from about250 g/ha to about 1200 g/ha, from about 280 g/ha to about 1120 g/ha andfrom about 560 g/ha to about 1120 g/ha. In particular embodiment, themonocotyledonous plant is selected from the group consisting of corn,rice, sorghum, wheat, rye, millet, sugarcane, oat, triticale,switchgrass, and turfgrass. Expressing the transgenic dicamba-degradingenzymatic activity such as a monooxygenase, in a monocotyledonous cropplant, such as corn, allows application of a higher level of dicamba tothe crop for the purpose of weed control at any stage of plant growth,as compared to the level of dicamba that may be applied to amonocotyledonous crop plant that does not comprise a transgene thatencodes such a dicamba-degrading enzymatic activity.

In yet another aspect, the invention provides a method for controllingweed growth in a field comprising: a) applying a herbicidally effectiveamount of an auxin-like herbicide other than dicamba to a field, whereinthe field comprises a transgenic dicotyledonous plant comprising anucleic acid encoding dicamba monooxygenase or is planted with a seedthat germinates into said transgenic dicotyledonous plant within 21 daysof applying the herbicide, wherein the herbicidally effective amount isan amount that does not damage the transgenic dicotyledonous plant butwill damage a plant of the same genotype that lacks the nucleic acidencoding dicamba monooxygenase, wherein the nucleic acid is selectedfrom the group consisting of (1) a nucleic acid sequence encoding thepolypeptide of SEQ ID NO:8, (2) a nucleic acid sequence comprising thesequence of SEQ ID NO:7, (3) a nucleic acid sequence that hybridizes toa complement of the nucleic acid sequence of SEQ ID NO:7 underconditions of 5×SSC, 50% formamide and 42° C., (4) a nucleic acidsequence having at least 70% sequence identity to the nucleic acidsequence of SEQ ID NO:7, and (5) a nucleic acid sequence encoding apolypeptide having at least 70% sequence identity to the polypeptidesequence of SEQ ID NO:8; and b) allowing the transgenic dicotyledonousplant to grow. In the method, step a) may comprise applying theherbicidally effective amount of an auxin-like herbicide to a growingenvironment adjacent to a growing environment comprising the transgenicdicotyledonous plant and allowing the herbicide to drift onto the plantor soil in which the plant grows. The auxin-like herbicide may be anyherbicide as described herein. In the method, step b) may compriseallowing the transgenic dicotyledonous plant to grow to maturity. Inspecific embodiments, the herbicidally effective amount may be definedas an amount that does not damage the transgenic plant.

In yet another aspect, the invention provides a method for increasingthe efficiency of use of a herbicide delivery device comprising: a)obtaining a device that has been used to deliver a first compositioncomprising an auxin-like herbicide; and b) delivering a secondcomposition to the field using the device without first completelywashing the device so that a herbicide residue comprising the auxin-likeherbicide remains in the device and is delivered with the secondcomposition to the field, wherein the field comprises a transgenicdicotyledonous plant expressing a nucleic acid encoding dicambamonooxygenase or is planted with a seed that germinates into saidtransgenic dicotyledonous plant within 21 days of delivering the secondcomposition, wherein the herbicide residue is present in an amount thatdoes not damage the transgenic dicotyledonous plant but will damage aplant of the same genotype that lacks the nucleic acid encoding dicambamonooxygenase, wherein the nucleic acid is selected from the groupconsisting of (1) a nucleic acid sequence encoding the polypeptide ofSEQ ID NO:8, (2) a nucleic acid sequence comprising the sequence of SEQID NO:7, (3) a nucleic acid sequence that hybridizes to a complement ofthe nucleic acid sequence of SEQ ID NO:7 under conditions of 5×SSC, 50%formamide and 42° C., (4) a nucleic acid sequence having at least 70%sequence identity to the nucleic acid sequence of SEQ ID NO:7, and (5) anucleic acid sequence encoding a polypeptide having at least 70%sequence identity to the polypeptide sequence of SEQ ID NO:8.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates, in one aspect, to the unexpected discovery thatpre-emergent applications of auxin-like herbicides such as dicamba maybe made close to, or even concurrently with the planting of crops. Theinvention provides superior weed control options, including reductionand/or prevention of herbicide tolerance in weeds. Pre-emergentapplications of auxin-like herbicides such as dicamba have previouslyrequired herbicide applications well in advance of planting andgermination of plants susceptible to auxin-like herbicides to allowbreakdown of the herbicide in the environment and avoid significant cropdamage or death. Most crop plants, and particularly dicotyledonousplants such as soybeans and cotton are extremely sensitive to dicamba.Thus, the recommended post-application delays in planting bymanufacturers must be closely followed.

Young plantlets and seeds are particularly sensitive to herbicides. Evenin transgenic seeds and plants, immature tissues can insufficientlyexpress the gene needed to render them tolerant to the herbicide, or maynot have accumulated sufficient levels of the protein to confertolerance. For example, mature plants have been found exhibiting highlevels of tolerance to the herbicides Harness™ (acetochlor), Lasso™(alachlor), Treflan™ (Trifluralin), Eptam™ (EPTC), and/or Far-Go™(triallate;//pmep.cce.cornell.edu/profiles/herb-growthreg/sethoxydim-vernolate/triallate/herb-prof-triallate.html)but susceptibility to the herbicides at germination. As a result of thisvariability in young tissues, crop response to post-emergenceapplications (e.g., in more mature vegetative tissues) of dicambaherbicides can significantly differ from the crop response topre-emergent applications of herbicides in which younger more sensitivetissues are exposed. The former does not necessarily predict the latter.This is underscored in the case of plants highly sensitive to a givenherbicide, such as dicots and the herbicide dicamba. Thus, the presentinvention unexpectedly shows that higher than predicted levels of cropsafety can be achieved from pre-emergence applications of dicamba.

The present invention employs auxin-like herbicides, which are alsocalled auxinic or growth regulator herbicides, or Group 4 herbicides(based on their mode of action). These types of herbicides mimic or actlike the natural plant growth regulators called auxins. The action ofauxinic herbicides appears to affect cell wall plasticity and nucleicacid metabolism, which can lead to uncontrolled cell division andgrowth.

Auxin-like herbicides include four chemical families: phenoxy,carboxylic acid (or pyridine), benzoic acid, and quinaline carboxylicacid. Phenoxy herbicides are most common and have been used asherbicides since the 1940s when (2,4-dichlorophenoxy)acetic acid (2,4-D)was discovered. Other examples include 4-(2,4-dichlorophenoxy)butyricacid (2,4-DB), 2-(2,4-dichlorophenoxy)propanoic acid (2,4-DP),(2,4,5-trichlorophenoxy)acetic acid (2,4,5-T),2-(2,4,5-Trichlorophenoxy)Propionic Acid (2,4,5-TP),2-(2,4-dichloro-3-methylphenoxy)-N-phenylpropanamide (clomeprop),(4-chloro-2-methylphenoxy)acetic acid (MCPA),4-(4-chloro-o-tolyloxy)butyric acid (MCPB), and2-(4-chloro-2-methylphenoxy)propanoic acid (MCPP).

The next largest chemical family is the carboxylic acid herbicides, alsocalled pyridine herbicides. Examples include3,6-dichloro-2-pyridinecarboxylic acid (Clopyralid),4-amino-3,5,6-trichloro-2-pyridinecarboxylic acid (picloram),(2,4,5-trichlorophenoxy) acetic acid (triclopyr), and4-amino-3,5-dichloro-6-fluoro-2-pyridyloxyacetic acid (fluroxypyr).Examples of benzoic acids include 3,6-dichloro-o-anisic acid (dicamba)and 3-amino-2,5-dichlorobenzoic acid (choramben). Dicamba is aparticularly useful herbicide for use in the present invention. A fourthchemical family of auxinic herbicides is the quinaline carboxylic acidfamily. Example includes 3,7-dichloro-8-quinolinecarboxylic acid(quinclorac). This herbicide is unique in that it also will control somegrass weeds, unlike the other auxin-like herbicides which essentiallycontrol only broadleaf or dicotyledonous plants. The other herbicide inthis category is 7-chloro-3-methyl-8-quinolinecarboxylic acid(quinmerac).

It was found, for example, that soybean plants transformed with dicambamonooxygenase (DMO)-encoding polynucleotide constructs were tolerant toeven early pre-emergence application of dicamba, with less than 10%injury rates at even 9× the labeled application rate (5,040 g/ha, 4.5lb/acre; Table 1). The inventors found that, even using an 18×application rate of 10,080 g/ha (9 lb/acre), injury to transgenicdicamba tolerant plants was less than 20% (Table 4). At an approximately2× rate of application of 1122 g/ha, less than 2% injury was observed.It was therefore indicated the improved weed control associated withpre- and post-emergence applications of herbicides may be used withoutany significant decreases in productivity due to herbicide damage.Pre-emergent applications of dicamba according to the invention maytherefore be combined with one or more herbicide applicationspost-emergence to dicamba-tolerant plants, while maintaining crop yieldand obtaining improved weed control. For example, one such herbicideapplication regime involved a late pre-emergence application of dicambain conjunction with a post-emergence application of dicamba at the V2stage of development. In certain embodiments, the post-emergenceapplication may be carried out at any point from emergence to harvest.Particularly beneficial will be post-emergence application at any Vstage until the soybean canopy closes, for example, at about the V1, V2,V3, V4, V5, V6 and/or later stages.

In accordance with the invention, methods and compositions for thecontrol of weeds are provided comprising the use of plants exhibitingtolerance to glyphosate and auxin-like herbicides such as dicamba. Asshown in the working examples, dicamba and glyphosate allow use ofdecreased amounts of herbicide to achieve the same level of control ofglyphosate-tolerant weeds and thus this embodiment provides asignificant advance for the control of herbicide tolerance in commercialproduction fields. In one embodiment, a tank mix of glyphosate anddicamba is applied pre- and/or post-emergence to plants. Glyphosate anddicamba may additionally be applied separately. In order to obtain theability to use decreased amount of herbicide, the glyphosate and dicambaare preferably applied within a sufficient interval that both herbicidesremain active and able to control weed growth.

This embodiment therefore allows use of lower amounts of eitherherbicide to achieve the same degree of weed control as an applicationof only one of the herbicides. For example, the invention providesmethods of weed control comprising applying in a field planted withtransgenic plants having tolerance to dicamba and glyphosate a herbicidecomposition comprising less than a 1× rate of glyphosate and/or dicamba,relative to the standard manufacturer labeled rate. Examples ofrespective glyphosate and dicamba application rates include from about a0.5×-0.95× of either herbicide, specifically including about 0.5×, 0.6×,0.7×, 0.8×. 0.85×, 0.9×, and 0.95× of either herbicide and all derivablecombinations thereof, as well as higher rates such as 0.97× and 0.99×.Alternatively, in the case of more difficult to control weeds or where agreater degree of weed control is desired, 1× and higher applicationrates may be made in view of the finding herein that even highapplication rates of dicamba did not significantly damage plants. The 1×application rates are set by the manufacturer of a commerciallyavailable herbicide formulation and are known to those of skill in theart. For example, the label for Fallow Master™, a glyphosate and dicambamixture having a ratio of glyphosate:dicamba of about 2:1 recommendsapplication rates of about 451 g/ha (311 ae g/ha glyphosate:140 ae g/hadicamba) to 621 ae g/ha (428 ae g/ha glyphosate: 193 ae g/ha dicamba)depending upon the weed species and weed height.

“Glyphosate” refers to N-phosphonomethylglycine and salts thereof.Glyphosate is commercially available in numerous formulations. Examplesof these formulations of glyphosate include, without limitation, thosesold by Monsanto Company as ROUNDUP®, ROUNDUP® ULTRA, ROUNDUP® ULTRAMAX,ROUNDUP® CT, ROUNDUP® EXTRA, ROUNDUP® BIACTIVE, ROUNDUP® BIOFORCE,RODEO®, POLARIS®, SPARK® and ACCORD® herbicides, all of which containglyphosate as its isopropylammonium salt, ROUNDUP® WEATHERMAX containingglyphosate as its potassium salt; ROUNDUP® DRY and RIVAL® herbicides,which contain glyphosate as its ammonium salt; ROUNDUP® GEOFORCE, whichcontains glyphosate as its sodium salt; and TOUCHDOWN® herbicide, whichcontains glyphosate as its trimethylsulfonium salt. “Dicamba” refers to3,6-dichloro-o-anisic acid or 3,6-dichloro-2-methoxy benzoic acid andits acids and salts. Its salts include isopropylamine, diglycoamine,dimethylamine, potassium and sodium. Examples of commercial formulationsof dicamba include, without limitation, Banvel™ (as DMA salt), Clarity™(as DGA salt), VEL-58-CS-11™ and Vanquish™ (as DGA salt, BASF).

Non-limiting examples of weeds that can be effectively controlled usingdicamba are the following: cheese weed, chick weed, while clover,cocklebur, Asiatic dayflower, deadnettle, red stem filaree, Carolinageranium, hemp sesbania, henbit, field horsetail (marestail), knotweed,kochia, lambsquarter, morningglory, indian mustard, wild mustard,redroot pigweed, smooth pigweed, prickly sida, cutleaf evening primrose,common purslane, common ragweed, gaint ragweed, russian thistle,shepardspurse, pennsylvania smartweed, spurge, velvetleaf, field violet,wild buckwheat, wild radish, soybeanpurslane, sicklepod, morninglory,wild buckwheat, common ragweed, horseweed (marestail), hairy fleabane,buckhorn plantain, and palmer pigweed. Non-limiting examples of weedsthat can be controlled using dicamba and glyphosate are the following:barnyardgrass, downy brome, volunteer cereals, Persian darnel, fieldsandbur, green foxtail, wild oats, wild buckwheat, volunteer canola,cowcockle, flixweed, kochia, ladysthumb, lambsquarters, wild mustard,prickly lettuce, redroot pigweed, smartweed, stinkgrass, stinkweed,russian thistle, foxtail, and witchgrass. Combining glyphosate anddicamba achieves the same level of weed control with reduced herbicideamounts and thus the spectrum of weeds that may be controlled at anygiven herbicide application rate may be increased when the herbicidesare combined.

Transgenic plants having herbicide tolerance may be made as described inthe art. Dicamba tolerance may be conferred, for example, by a gene fordicamba monooxygenase (DMO) from Pseudomonas maltophilia (US PatentApplication No: 20030135879). Examples of sequences that may be used inthis regard are nucleic acid encoding the polypeptides of SEQ ID Nos: 2,4, 6, 8, 10, and 12. Examples of sequences encoding these polypeptidesare given as SEQ ID NOS: 1, 3, 5, 7, 9, and 11. SEQ ID NO: 1 shows DMOfrom Pseudomonas maltophilia optimized for expression in dicots usingArabidopsis thaliana codon usage. The polypeptide, predicted to have anAla, Thr, Cys at positions 2, 3, 112, respectively, is given in SEQ IDNO:2. SEQ ID NO:3 shows another Pseudomonas maltophilia DMO optimizedfor expression in dicots and encoding the polypeptide of SEQ ID NO:4,predicted to have an Leu, Thr, Cys at positions 2, 3, 112, respectively.SEQ ID NO:5 shows the coding sequence and SEQ ID NO:6 the polypeptidefor dicot optimized DMO predicted to have a Leu, Thr, Trp at positions2, 3, 112, respectively. SEQ ID NOS:7 and 8 show the coding andpolypeptide sequences for DMO predicted to have an Ala, Thr, Cys atposition 2, 3, 112, respectively. SEQ ID NOS:9 and 10 show thedicot-optimized coding sequence and polypeptide sequences for DMOpredicted to have an Ala, Thr, Trp at positions 2, 3, 112, respectively.SEQ ID NOS:11 and 12 show coding sequence and polypeptide sequences forDMO from Pseudomonas maltophilia (US Patent Application No:20030135879). Another exemplary DMO sequence may be a DMO predicted tohave a Leu, Thr, Cys at position 2, 3, 112, respectively with codonusage of Pseudomonas maltophilia (US Patent Application No:20030135879).

Sequences conferring glyphosate tolerance are also known, includingglyphosate resistant 5-enolpyruvylshikimate-3-phosphate synthases(EPSPS) as described in U.S. Pat. No. 5,627,061, U.S. Pat. No.5,633,435, U.S. Pat. No. 6,040,497, U.S. Pat. No. 5,094,945, WO04074443,WO04009761, all of which are hereby incorporated by reference; byexpression of nucleic acids encoding glyphosate degrading enzymes, forexample, glyphosate oxidoreductase (GOX, U.S. Pat. No. 5,463,175, hereinincorporated by reference), glyphosate decarboxylase (WO05003362; USPatent Application 20040177399, herein incorporated by reference); andby expression of nucleic acids encoding glyphosate inactivating enzymes,such as glyphosate-N-acetyl transferase (GAT, e.g. U.S. Patentpublications 20030083480 and 20070079393, herein incorporated byreference).

Variants of proteins having a capability to degrade auxin-likeherbicides, glyphosate or other herbicides can readily be prepared andassayed for activity according to standard methods. Such sequences canalso be identified by techniques know in the art, for example, fromsuitable organisms including bacteria that degrade auxin-like herbicidessuch as dicamba or other herbicides (U.S. Pat. No. 5,445,962; Cork andKrueger, 1991; Cork and Khalil, 1995). One means of isolating a DMO orother sequence is by nucleic acid hybridization, for example, to alibrary constructed from the source organism, or by RT-PCR using mRNAfrom the source organism and primers based on the disclosed desaturases.The invention therefore encompasses use of nucleic acids hybridizingunder stringent conditions to a DMO encoding sequence described herein.One of skill in the art understands that conditions may be rendered lessstringent by increasing salt concentration and decreasing temperature.Thus, hybridization conditions can be readily manipulated, and thus willgenerally be a method of choice depending on the desired results. Anexample of high stringency conditions is 5×SSC, 50% formamide and 42° C.By conducting a wash under such conditions, for example, for 10 minutes,those sequences not hybridizing to a particular target sequence underthese conditions can be removed.

Variants can also be chemically synthesized, for example, using theknown DMO polynucleotide sequences according to techniques well known inthe art. For instance, DNA sequences may be synthesized byphosphoamidite chemistry in an automated DNA synthesizer. Chemicalsynthesis has a number of advantages. In particular, chemical synthesisis desirable because codons preferred by the host in which the DNAsequence will be expressed may be used to optimize expression. Not allof the codons need to be altered to obtain improved expression, butpreferably at least the codons rarely used in the host are changed tohost-preferred codons. High levels of expression can be obtained bychanging greater than about 50%, most preferably at least about 80%, ofthe codons to host-preferred codons. The codon preferences of many hostcells are known (PCT WO 97/31115; PCT WO 97/11086; EP 646643; EP 553494;and U.S. Pat. Nos. 5,689,052; 5,567,862; 5,567,600; 5,552,299 and5,017,692). The codon preferences of other host cells can be deduced bymethods known in the art. Also, using chemical synthesis, the sequenceof the DNA molecule or its encoded protein can be readily changed to,for example, optimize expression (for example, eliminate mRNA secondarystructures that interfere with transcription or translation), add uniquerestriction sites at convenient points, and delete protease cleavagesites.

Modification and changes may be made to the polypeptide sequence of aprotein such as the DMO sequences provided herein while retainingenzymatic activity. The following is a discussion based upon changingthe amino acids of a protein to create an equivalent, or even animproved, modified polypeptide and corresponding coding sequences. It isknown, for example, that certain amino acids may be substituted forother amino acids in a protein structure without appreciable loss ofinteractive binding capacity with structures such as binding sites onsubstrate molecules. Since it is the interactive capacity and nature ofa protein that defines that protein's biological functional activity,certain amino acid sequence substitutions can be made in a proteinsequence, and, of course, its underlying DNA coding sequence, andnevertheless obtain a protein with like properties. It is thuscontemplated that various changes may be made in the DMO peptidesequences described herein or other herbicide tolerance polypeptides andcorresponding DNA coding sequences without appreciable loss of theirbiological utility or activity.

In making such changes, the hydropathic index of amino acids may beconsidered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a protein is generallyunderstood in the art (Kyte et al., 1982). It is accepted that therelative hydropathic character of the amino acid contributes to thesecondary structure of the resultant protein, which in turn defines theinteraction of the protein with other molecules, for example, enzymes,substrates, receptors, DNA, antibodies, antigens, and the like. Eachamino acid has been assigned a hydropathic index on the basis of theirhydrophobicity and charge characteristics (Kyte et al., 1982), theseare: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine(+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8);glycine (−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9);tyrosine (−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5);glutamine (−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9);and arginine (−4.5).

It is known in the art that amino acids may be substituted by otheramino acids having a similar hydropathic index or score and still resultin a protein with similar biological activity, i.e., still obtain abiological functionally equivalent protein. In making such changes, thesubstitution of amino acids whose hydropathic indices are within ±2 ispreferred, those which are within ±1 are particularly preferred, andthose within ±0.5 are even more particularly preferred.

It is also understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity. U.S. Pat.No. 4,554,101 states that the greatest local average hydrophilicity of aprotein, as governed by the hydrophilicity of its adjacent amino acids,correlates with a biological property of the protein. As detailed inU.S. Pat. No. 4,554,101, the following hydrophilicity values have beenassigned to amino acid residues: arginine (+3.0); lysine (+3.0);aspartate (+3.0±1); glutamate (+3.0 ±1); serine (+0.3); asparagine(+0.2); glutamine (+0.2); glycine (0); threonine (−0.4); proline (−0.5±1); alanine (−0.5); histidine (−0.5); cysteine (−1.0); methionine(−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8); tyrosine(−2.3); phenylalanine (−2.5); tryptophan (−3.4). It is understood thatan amino acid can be substituted for another having a similarhydrophilicity value and still obtain a biologically equivalent protein.In such changes, the substitution of amino acids whose hydrophilicityvalues are within ±2 is preferred, those which are within ±1 areparticularly preferred, and those within ±0.5 are even more particularlypreferred. Exemplary substitutions which take these and various of theforegoing characteristics into consideration are well known to those ofskill in the art and include: arginine and lysine; glutamate andaspartate; serine and threonine; glutamine and asparagine; and valine,leucine and isoleucine.

A gene conferring herbicide tolerance will typically be linked to aplant promoter driving expression of the gene in an amount sufficient toconfer the herbicide tolerance. Promoters suitable for this and otheruses are well known in the art. Examples describing such promotersinclude U.S. Pat. No. 6,437,217 (maize RS81 promoter), U.S. Pat. No.5,641,876 (rice actin promoter), U.S. Pat. No. 6,426,446 (maize RS324promoter), U.S. Pat. No. 6,429,362 (maize PR-1 promoter), U.S. Pat. No.6,232,526 (maize A3 promoter), U.S. Pat. No. 6,177,611 (constitutivemaize promoters), U.S. Pat. Nos. 5,322,938, 5,352,605, 5,359,142 and5,530,196 (35S promoter), U.S. Pat. No. 6,433,252 (maize L3 oleosinpromoter), U.S. Pat. No. 6,429,357 (rice actin 2 promoter as well as arice actin 2 intron), U.S. Pat. No. 5,837,848 (root specific promoter),U.S. Pat. No. 6,294,714 (light inducible promoters), U.S. Pat. No.6,140,078 (salt inducible promoters), U.S. Pat. No. 6,252,138 (pathogeninducible promoters), U.S. Pat. No. 6,175,060 (phosphorus deficiencyinducible promoters), U.S. Pat. No. 6,388,170 (PC1SV promoter), U.S.Pat. No. 6,635,806 (gamma-coixin promoter), and U.S. patent applicationSer. No. 09/757,089 (maize chloroplast aldolase promoter). Additionalpromoters that may find use are a nopaline synthase (NOS) promoter(Ebert et al., 1987), the octopine synthase (OCS) promoter (which iscarried on tumor-inducing plasmids of Agrobacterium tumefaciens), thecaulimovirus promoters such as the cauliflower mosaic virus (CaMV) 19Spromoter (Lawton et al., 1987), the CaMV 35S promoter (Odell et al.,1985), the figwort mosaic virus 35S-promoter (Walker et al., 1987), thesucrose synthase promoter (Yang et al., 1990), the R gene complexpromoter (Chandler et al., 1989), the chlorophyll a/b binding proteingene promoter, CaMV35S (U.S. Pat. Nos. 5,322,938; 5,352,605; 5,359,142;and 5,530,196), FMV35S (U.S. Pat. Nos. 6,051,753; 5,378,619), a PC1SVpromoter (U.S. Pat. No. 5,850,019; or SEQ ID NO:20), the SCP promoter(U.S. Pat. No. 6,677,503), and AGRtu.nos (GenBank Accession V00087;Depicker et al, 1982; Bevan et al., 1983) promoters, and the like (seealso see Table 1).

Benefit may be obtained for the expression of herbicide tolerance genesby use of a sequence coding for a transit peptide. For example,incorporation of a suitable chloroplast transit peptide, such as, theArabidopsis thaliana EPSPS CTP (Klee et al., 1987), and the Petuniahybrida EPSPS CTP (della-Cioppa et al., 1986) has been shown to targetheterologous EPSPS protein sequences to chloroplasts in transgenicplants. Chloroplast transit peptides (CTPs) are engineered to be fusedto the N-terminus of a protein to direct the protein into the plantchloroplast. Such sequences may find use in connection with a nucleicacid conferring dicamba tolerance in particular. Manychloroplast-localized proteins are expressed from nuclear genes asprecursors and are targeted to the chloroplast by a chloroplast transitpeptide that is removed during the import process. Examples ofchloroplast proteins include the small subunit (RbcS2) ofribulose-1,5,-bisphosphate carboxylase, ferredoxin, ferredoxinoxidoreductase, the light-harvesting complex protein I and protein II,and thioredoxin F. Other exemplary chloroplast targeting sequencesinclude the maize cab-m7 signal sequence (Becker et al., 1992; PCT WO97/41228), the pea glutathione reductase signal sequence (Creissen etal., 1995; PCT WO 97/41228), and the CTP of the Nicotiana tabacumribulose 1,5-bisphosphate carboxylase small subunit chloroplast transitpeptide (SSU-CTP) (Mazur, et al., 1985). Use of AtRbcS4 (CTP1; U.S. Pat.No. 5,728,925), AtShkG (CTP2; Klee et al., 1987), AtShkGZm(CTP2synthetic; see SEQ ID NO:14 of WO04009761), and PsRbcS (Coruzzi etal., 1984), as well as others disclosed, for instance, in U.S.Provisional Patent Application 60/891,675, peptide and nucleic acidsequences for which are listed herein at SEQ ID NOs:21-32, may be ofbenefit for use with the invention.

A 5′ UTR that functions as a translation leader sequence is a DNAgenetic element located between the promoter sequence of a gene and thecoding sequence. The translation leader sequence is present in the fullyprocessed mRNA upstream of the translation start sequence. Thetranslation leader sequence may affect processing of the primarytranscript to mRNA, mRNA stability or translation efficiency. Examplesof translation leader sequences include maize and petunia heat shockprotein leaders (U.S. Pat. No. 5,362,865), plant virus coat proteinleaders, plant rubisco leaders, among others (Turner and Foster, 1995).Non-limiting examples of 5′ UTRs that may in particular be of benefitfor use GmHsp (U.S. Pat. No. 5,659,122), PhDnaK (U.S. Pat. No.5,362,865), AtAnt1, TEV (Carrington and Freed, 1990), and AGRtunos(GenBank Accession V00087; Bevan et al., 1983).

The 3′ non-translated sequence, 3′ transcription termination region, orpoly adenylation region means a DNA molecule linked to and locateddownstream of a structural polynucleotide molecule and includespolynucleotides that provide polyadenylation signal and other regulatorysignals capable of affecting transcription, mRNA processing or geneexpression. The polyadenylation signal functions in plants to cause theaddition of polyadenylate nucleotides to the 3′ end of the mRNAprecursor. The polyadenylation sequence can be derived from the naturalgene, from a variety of plant genes, or from T-DNA genes. An example ofa 3′ transcription termination region is the nopaline synthase 3′ region(nos 3′; Fraley et al., 1983). The use of different 3′ nontranslatedregions is exemplified (Ingelbrecht et al., 1989). Polyadenylationmolecules from a Pisum sativum RbcS2 gene (Ps.RbcS2-E9; Coruzzi et al.,1984) and AGRtu.nos (Rojiyaa et al., 1987, Genbank Accession E01312) inparticular may be of benefit for use with the invention.

Intron sequences are known in the art to aid in the expression oftransgenes in monocot plant cells. Examples of introns include the cornactin intron (U.S. Pat. No. 5,641,876), the corn HSP70 intron (ZmHSP70;U.S. Pat. No. 5,859,347; U.S. Pat. No. 5,424,412), and rice TPI intron(OsTPI; U.S. Pat. No. 7,132,528), and are of benefit in practicing thisinvention.

Any of the techniques known in the art for introduction of transgenesinto plants may be used to prepare a herbicide tolerant plant inaccordance with the invention (see, for example, Mild et al., 1993).Suitable methods for transformation of plants are believed to includevirtually any method by which DNA can be introduced into a cell, such asby electroporation as illustrated in U.S. Pat. No. 5,384,253;microprojectile bombardment as illustrated in U.S. Pat. Nos. 5,015,580;5,550,318; 5,538,880; 6,160,208; 6,399,861; and 6,403,865;Agrobacterium-mediated transformation as illustrated in U.S. Pat. Nos.5,635,055; 5,824,877; 5,591,616; 5,981,840; and 6,384,301; andprotoplast transformation as illustrated in U.S. Pat. No. 5,508,184,etc. Through the application of techniques such as these, the cells ofvirtually any plant species may be stably transformed, and these cellsdeveloped into transgenic plants. Techniques that may be particularlyuseful in the context of cotton transformation are disclosed in U.S.Pat. Nos. 5,846.797, 5,159,135, 5,004,863, and 6,624,344; and techniquesfor transforming Brassica plants in particular are disclosed, forexample, in U.S. Pat. No. 5,750,871; and techniques for transformingsoybean are disclosed in for example in Zhang et al., 1999 and U.S. Pat.No. 6,384,301). Corn can be transformed using methods described inWO9506722 and US patent application 20040244075.

After effecting delivery of exogenous DNA to recipient cells, the nextsteps generally concern identifying the transformed cells for furtherculturing and plant regeneration. In order to improve the ability toidentify transformants, one may desire to employ a selectable orscreenable marker gene with a transformation vector prepared inaccordance with the invention. In this case, one would then generallyassay the potentially transformed cell population by exposing the cellsto a selective agent or agents, or one would screen the cells for thedesired marker gene trait.

Cells that survive the exposure to the selective agent, or cells thathave been scored positive in a screening assay, may be cultured in mediathat supports regeneration of plants. In an exemplary embodiment, anysuitable plant tissue culture media, for example, MS and N6 media may bemodified by including further substances such as growth regulators.Tissue may be maintained on a basic media with growth regulators untilsufficient tissue is available to begin plant regeneration efforts, orfollowing repeated rounds of manual selection, until the morphology ofthe tissue is suitable for regeneration, typically at least 2 weeks,then transferred to media conducive to shoot formation. Cultures aretransferred periodically until sufficient shoot formation has occurred.Once shoot are formed, they are transferred to media conducive to rootformation. Once sufficient roots are formed, plants can be transferredto soil for further growth and maturity.

To confirm the presence of the exogenous DNA or “transgene(s)” in theregenerating plants, a variety of assays may be performed. Such assaysinclude, for example, “molecular biological” assays, such as Southernand Northern blotting and PCR™; “biochemical” assays, such as detectingthe presence of a protein product, e.g., by immunological means (ELISAsand Western blots) or by enzymatic function; plant part assays, such asleaf or root assays; and also, by analyzing the phenotype of the wholeregenerated plant.

Once a transgene has been introduced into a plant, that gene can beintroduced into any plant sexually compatible with the first plant bycrossing, without the need for ever directly transforming the secondplant. Therefore, as used herein the term “progeny” denotes theoffspring of any generation of a parent plant prepared in accordancewith the instant invention, wherein the progeny comprises a selected DNAconstruct prepared in accordance with the invention. A “transgenicplant” may thus be of any generation. “Crossing” a plant to provide aplant line having one or more added transgenes or alleles relative to astarting plant line, as disclosed herein, is defined as the techniquesthat result in a particular sequence being introduced into a plant lineby crossing a starting line with a donor plant line that comprises atransgene or allele of the invention. To achieve this one could, forexample, perform the following steps: (a) plant seeds of the first(starting line) and second (donor plant line that comprises a desiredtransgene or allele) parent plants; (b) grow the seeds of the first andsecond parent plants into plants that bear flowers; (c) pollinate aflower from the first parent plant with pollen from the second parentplant; and (d) harvest seeds produced on the parent plant bearing thefertilized flower.

The preparation of herbicide compositions for use in connection with thecurrent invention will be apparent to those of skill in the art in viewof the disclosure. Such compositions, which are commercially available,will typically include, in addition to the active ingredient, componentssuch as surfactants, solid or liquid carriers, solvents and binders.Examples of surfactants that may be used for application to plantsinclude the alkali metal, alkaline earth metal or ammonium salts ofaromatic sulfonic acids, e.g., ligno-, phenol-, naphthalene- anddibutylnaphthalenesulfonic acid, and of fatty acids of arylsulfonates,of alkyl ethers, of lauryl ethers, of fatty alcohol sulfates and offatty alcohol glycol ether sulfates, condensates of sulfonatednaphthalene and its derivatives with formaldehyde, condensates ofnaphthalene or of the naphthalenesulfonic acids with phenol andformaldehyde, condensates of phenol or phenolsulfonic acid withformaldehyde, condensates of phenol with formaldehyde and sodiumsulfite, polyoxyethylene octylphenyl ether, ethoxylated isooctyl-,octyl- or nonylphenol, tributylphenyl polyglycol ether, alkylarylpolyether alcohols, isotridecyl alcohol, ethoxylated castor oil,ethoxylated triarylphenols, salts of phosphatedtriarylphenolethoxylates, lauryl alcohol polyglycol ether acetate,sorbitol esters, lignin-sulfite waste liquors or methylcellulose, ormixtures of these. Common practice in the case of surfactant use isabout 0.25% to 1.0% by weight, and more commonly about 0.25% to 0.5% byweight.

Compositions for application to plants may be solid or liquid. Wheresolid compositions are used, it may be desired to include one or morecarrier materials with the active compound. Examples of carriers includemineral earths such as silicas, silica gels, silicates, talc, kaolin,attaclay, limestone, chalk, loess, clay, dolomite, diatomaceous earth,calcium sulfate, magnesium sulfate, magnesium oxide, ground syntheticmaterials, fertilizers such as ammonium sulfate, ammonium phosphate,ammonium nitrate, thiourea and urea, products of vegetable origin suchas cereal meals, tree bark meal, wood meal and nutshell meal, cellulosepowders, attapulgites, montmorillonites, mica, vermiculites, syntheticsilicas and synthetic calcium silicates, or mixtures of these.

For liquid solutions, water-soluble compounds or salts may be included,such as sodium sulfate, potassium sulfate, sodium chloride, potassiumchloride, sodium acetate, ammonium hydrogen sulfate, ammonium chloride,ammonium acetate, ammonium formate, ammonium oxalate, ammoniumcarbonate, ammonium hydrogen carbonate, ammonium thiosulfate, ammoniumhydrogen diphosphate, ammonium dihydrogen monophosphate, ammonium sodiumhydrogen phosphate, ammonium thiocyanate, ammonium sulfamate or ammoniumcarbamate.

Other exemplary components in herbicidal compositions include binderssuch as polyvinylpyrrolidone, polyvinyl alcohol, partially hydrolyzedpolyvinyl acetate, carboxymethylcellulose, starch,vinylpyrrolidone/vinyl acetate copolymers and polyvinyl acetate, ormixtures of these; lubricants such as magnesium stearate, sodiumstearate, talc or polyethylene glycol, or mixtures of these; antifoamssuch as silicone emulsions, long-chain alcohols, phosphoric esters,acetylene diols, fatty acids or organofluorine compounds, and complexingagents such as: salts of ethylenediaminetetraacetic acid (EDTA), saltsof trinitrilotriacetic acid or salts of polyphosphoric acids, ormixtures of these.

Equipment and methods known in the art are used to apply variousherbicide treatments as disclosed herein. The application rates ofherbicides maybe varied, for instance as described above, depending uponthe soil texture, pH, organic matter content, tillage systems, and thesize of the weed, and can be determined by consulting the herbicidelabel for the proper herbicide rate.

EXAMPLES

The following examples are included to illustrate embodiments of theinvention. It should be appreciated by those of skill in the art thatthe techniques disclosed in the examples that follow representtechniques discovered by the inventor to function well in the practiceof the invention. However, those of skill in the art should, in light ofthe present disclosure, appreciate that many changes can be made in thespecific embodiments which are disclosed and still obtain a like orsimilar result without departing from the concept, spirit and scope ofthe invention. More specifically, it will be apparent that certainagents which are both chemically and physiologically related may besubstituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

Example 1 Tolerance of Soybean Plants Containing DMO-EncodingPolynucleotide Construct to Early Pre-Emergence Application of Dicamba

Transgenic soybean plants were obtained by Agrobacterium transformationof soybean cotyledonary nodes using standard procedures and a binaryvector containing the DMO-encoding polynucleotide given as SEQ ID NO:7,which encodes the polypeptide of SEQ ID NO:8. Four transgenic soybeanevents were prepared and designated Events 1-4. Transgenic soybeanplants containing the events were tested for their tolerance to dicambaherbicide relative to controls, confirming herbicide tolerance.Non-transgenic soybean plants were used as controls.

Transgenic and control soybean seeds were planted into 3.5-inch squareplastic pots containing Redi-earth™ (Scotts-Siena Horticultural ProductsCo., Marysville, Ohio). The soil surface was treated with variousamounts (561 to 5040 g/ha, 0.5 to 4.5 lb/acre, or 1× to 9× labeledrates) of dicamba formulations (Clarity™ or Banvel™, BASF, Raleigh,N.C.). The pots were placed on capillary matting in 35 inch×60 inchfiberglass watering trays for overhead and/or sub-irrigation for theduration of the test period so as to maintain optimum soil moisture forplant growth and were fertilized with Osmocote (14-14-14 slow release;Scotts-Siena Horticultural Products Co., Marysville, Ohio) at the rateof 100 gm/cu.ft. to sustain plant growth for the duration of greenhousetrials.

The plants were grown in greenhouses at 27°/21° C. day/night temperaturewith relative humidity between 25%-75% to simulate warm season growingconditions of late spring. A 14 h minimum photoperiod was provided withsupplemental light at about 600 μE as needed. Trials were established ina randomized block design randomized by rate with 4 to 6 replications ofeach treatment depending on plant quality, availability, and to accountfor any environmental variability that may have occurred within theconfines of each greenhouse.

Treated plants in greenhouse trials were visually assessed at aparticular day after treatment (DAT) for injury on a scale of 0 to 100percent relative to untreated control plants, with zero representing“no” injury and 100% representing “complete” injury or death. Data werecollected and analyzed using suitable statistical methods.

The results of the study surprisingly showed that soybean plantstransformed with the DMO-encoding polynucleotide construct were tolerantto even early pre-emergence application of dicamba. As indicated inTable 1 below, injury to the transgenic plants was less than 10% even atthe highest application rate i.e., 5040 g/ha, 4.5 lb/acre, or 9× labeledrates of dicamba.

TABLE 1 Percentage injury to non-transgenic or transgenic soybean plantsfrom early pre-emergence application of dicamba at sowing. FormulationClarity ™ % injury at shown rates (g ae/ha*) at 14 DAT ID 561 840 22444485 5040 Control 67.0 a 73.0 b 96.6 a 98.2 a 99.5 a Control 61.0 a 86.0a 98.1 a 98.3 a 99.8 a Event 1 0.0 b 0.0 c 1.7 bc 0.7 b 3.1 b Event 20.0 b 0.0 c 1.1 c 1.0 b 2.2 b Event 3 0.0 b 0.0 c 1.1 c 0.6 b 3.5 bEvent 4 0.0 b 0.0 c 4.4 b 0.8 b 7.2 b LSD 9.9 7.2 3.2 2.2 5.1 The %injury was represented as ANOVA mean comparisons. Similar lettersrepresent no statistical difference at the p = 0.05 level.

Example 2 Tolerance of Soybean Plants Containing a DMO-EncodingPolynucleotide Construct to Early Pre-Emergence Application of Dicambaat Sowing Followed by Post-Emergence Application of Dicamba

In addition to the method described in Example 2 for early pre-emergence(at sowing) application of dicamba, post-emergence (V2 stage of soybeandevelopment) application of dicamba was made with a track sprayer usingthe Teejet 9501E flat fan nozzle (Spraying Systems Co, Wheaton, Ill.)with the air pressure set at a minimum of 24 psi (165 kpa). The spraynozzle was kept at a height of about 16 inches above the top of theplant material for spraying. The spray volume was 10 gallons per acre or93 liters per hectare.

As shown in Table 2, soybean plants transformed with the DMO-encodingpolynucleotide construct were tolerant to early pre-emergenceapplications of dicamba at sowing followed by post-emergence applicationof dicamba. Surprisingly, injury to transgenic plants was less than 20%at the overall dicamba rate of 10080 g/ha, 9 lb/acre or 18× labeledrate.

TABLE 2 Percentage injury to non-transgenic or transgenic soybean plantsfrom application of dicamba at sowing followed by post-emergenceapplication at V2 stage.* Formulation Clarity ™ % injury at shown rates(g ae/ha*) at 28 DAT Plants 1122 1680 4488 8970 10080 Control 97.5 a98.8 a 99.8 a 100.0 a 100.0 a Control 95.6 a 98.1 a 99.4 a 100.0 a 100.0a Event 1 0.0 c 1.8 b 4.5 d 11.9 c 16.9 b Event 2 2.6 bc 3.9 b 8.1 bc13.8 b 16.9 b Event 3 3.1 b 2.9 b 8.8 b 11.9 c 17.5 b Event 4 2.3 bc 2.0b 6.9 c 11.9 c 15.6 b LSD 3.1 2.2 1.4 1.6 1.9 *The % injury wasrepresented as ANOVA mean comparisons. Similar letters represent nostatistical difference at the p = 0.05 level.

Example 3 Tolerance of Soybean Plants Containing DMO-EncodingPolynucleotide Construct to Late Pre-Emergence Application of Dicamba

An analysis was carried out of the effect of late pre-emergenceapplications of dicamba at soil cracking due to emergence of soybeanseedling hypocotyls. Dicamba applications were made using a tracksprayer as described in the previous examples. As shown in Table 3,soybean plants transformed with the DMO-encoding polynucleotideconstruct were found to be tolerant to late pre-emergence application ofdicamba at soil cracking. Significantly, injury in the transgenic eventswas less than 5% even at the highest rate i.e., 5040 g/ha, 4.5 lb/acre,or 9× labeled rates of dicamba.

TABLE 3 Percentage injury to non-transgenic or transgenic soybean plantsfrom late pre-emergence application of dicamba at soil cracking.*Formulation Clarity ™ % injury at shown rates (g ae/ha*) at 14 DATPlants 561 840 2244 4485 5040 Control 86.9 a 96.8 a 98.4 A 98.5 a 99.2 aControl 89.6 a 91.9 a 98.4 A 99.0 a 99.4 a Event 1 0.0 b 0.0 b 0.5 C 2.5bc 2.0 b Event 2 0.0 b 0.0 b 2.9 bc 0.0 c 1.5 b Event 3 0.0 b 0.0 b 1.5bc 4.4 b 1.3 b Event 4 0.0 b 0.5 b 3.3 B 3.0 bc 1.3 b LSD 8.1 5.4 2.43.9 2.3 *The % injury was represented as ANOVA mean comparisons. Similarletters represent no statistical difference at the p = 0.05 level.

Example 4 Tolerance of Soybean Plants Containing DMO-EncodingPolynucleotide Construct to Late Pre-Emergence Applications of DicambaFollowed by Post-Emergence Applications of Dicamba

In addition to the studies above, an analysis was carried out of theeffect of late pre-emergence applications of dicamba at soil crackingfollowed by post-emergence application of dicamba at the V2 stage ofdevelopment. As shown in Table 4, soybean plants transformed with theDMO-encoding polynucleotide construct were tolerant to latepre-emergence application of dicamba at soil cracking and post-emergenceapplication of dicamba. Injury to transgenic events was less than 20%even at the overall dicamba rate of 10080 g/ha, 9 lb/acre, or 18×labeled rate.

TABLE 4 Percentage injury to non-transgenic or transgenic soybean plantsfrom late pre-emergence application of dicamba at soil cracking followedby post-emergence application at V2 stage.* Formulation Clarity ™ %injury at shown rates (g ae/ha*) at 28 DAT Plants 1122 1680 4488 897010080 Control 95.6 a 98.1 a 100.0 a 100.0 a 100.0 a Control 95.0 a 98.1a 99.4 a 99.8 a 100.0 a Event 1 0.3 b 0.9 b 6.3 b 13.1 b 16.3 bc Event 20.8 b 1.6 b 6.0 b 11.3 c 15.0 c Event 3 1.0 b 1.4 b 7.5 b 11.3 c 17.5 bEvent 4 1.8 b 1.8 b 7.5 b 13.1 b 16.3 bc LSD 4.5 2.7 1.6 1.6 1.9 *The %injury was represented as ANOVA mean comparisons. Similar lettersrepresent no statistical difference at the p = 0.05 level.

Example 5 Tolerance of Soybean Plants Containing DMO-EncodingPolynucleotide Construct to Pre- and Post-Emergence Application ofDicamba in the Field

Non-transgenic and transgenic soybean seeds were planted around thebeginning of the growing season at the time of optimum growth conditionsdepending on soil moisture, temperature, and seeding depth. Across alllocations seeds were planted under split-plot design with dicambatreatments as whole-plot effects and events as split-plot effects. Thedesign details were as follows: 6 locations, 2 replications/location, 2rows/plot, row length 12 feet (+3 ft alley), 9 seeds/foot, 108seeds/row, 5 events (Events 1-4 and a fifth event that was segregating);and 4 treatments as shown below in Table 5. In all 240 plots wereplanted at 6 locations (40 per location).

TABLE 5 Details of 4 treatments applied to show the tolerance oftransgenic soybean to dicamba. 1st Application 2nd Application TreatmentRate Plant Stage Rate Plant Stage 1 NO Dicamba NO Dicamba NO Dicamba NODicamba 2 1.5 lb ae/acre At Planting N/A N/A 3 N/A N/A 1.5 lb ae/acreV3-4 4 1.5 lb ae/acre At Planting 1.5 lb ae/acre V3-4

Four non-transgenic border rows were planted all around the trial usinga known commercial line such as A3525. Optimum production and managementpractices known in the art were followed. Maximum pest control anddisease control was practiced as needed to prevent confounding effectsof dicamba applications. The field was irrigated as needed according tostandard practices.

All plants in the field were treated with pre-emergence andpost-emergence applications of dicamba and visually assessed at aparticular day after planting for injury on a scale of 0 to 100 percentrelative to untreated control plants, with zero representing “no” injuryand 100% representing “complete” injury or death. Seed planting andpre-emergence treatment were carried out approximately one-month apartin late spring in Monmouth, Ill. As shown in Table 6, it was found thatall transgenic soybean plants had no or very little injury. A fifthtransgenic event used appeared to be segregating, so a certainpercentage of plants died after the treatments.

TABLE 6 Tolerance of soybean plants containing DMO-encodingpolynucleotide construct to pre- and post-emergence application ofdicamba in field.* Dead % Inj % Inj % Inj % Inj % GR % Inj % GR or Event# Trmt 6/7 6/13 6/20 6/27 6/27 7/5 7/5 Stunted 1 No spray 0 0 0 0 0 2 00 1 No spray 0 0 0 0 0 0 0 0 2 No spray 0 0 0 1 0 3 3 0 2 No spray 0 0 00 0 0 0 0 3 No spray 0 0 0 1 0 3 0 0 3 No spray 0 0 0 0 0 3 0 0 4 Nospray 0 0 0 2 0 0 0 0 4 No spray 0 0 0 0 0 5 0 0 5 No spray 0 0 0 7 0 52 0 5 No spray 0 0 0 7 3 7 3 0 1 Pre at sowing 0 0 0 1 0 0 0 0 1 Pre atsowing 0 2 0 2 0 0 0 0 2 Pre at sowing 0 0 0 5 0 0 0 0 2 Pre at sowing 00 0 2 0 0 0 0 3 Pre at sowing 0 4 0 1 0 0 0 0 3 Pre at sowing 0 2 0 5 03 0 0 4 Pre at sowing 0 3 0 5 0 0 0 0 4 Pre at sowing 0 4 0 2 0 0 0 0 5Pre at sowing 0 15 15 5 0 0 0 24 5 Pre at sowing 0 8 10 2 0 0 0 14 1Post at V3 0 0 0 5 0 0 0 0 1 Post at V3 0 0 0 7 0 0 0 2 2 Post at V3 0 00 3 0 0 0 0 2 Post at V3 0 0 0 3 0 2 0 1 3 Post at V3 0 0 0 3 3 0 0 0 3Post at V3 0 0 0 5 0 3 0 0 4 Post at V3 0 0 0 3 0 0 0 0 4 Post at V3 0 00 3 0 0 0 0 5 Post at V3 0 0 0 7 0 2 0 15 5 Post at V3 0 0 0 5 5 2 0 151 Pre & Post 0 0 0 5 0 0 0 3 1 Pre & Post 0 2 2 5 3 0 0 0 2 Pre & Post 00 0 1 0 2 0 0 2 Pre & Post 0 0 0 2 0 0 0 0 3 Pre & Post 0 0 0 3 0 2 0 03 Pre & Post 0 2 0 3 0 0 0 0 4 Pre & Post 0 0 0 3 8 2 2 0 4 Pre & Post 01 0 3 3 0 0 0 5 Pre & Post 0 15 10 3 5 0 0 23 5 Pre & Post 0 10 10 1 0 00 20 *No spray means no dicamba was applied to the plants. Pre at sowingmeans 1.5 lb/acre of dicamba was applied at planting. Post at V3 means1.5 lb/acre of dicamba was applied 4 weeks after planting. Pre and postmeans 1.5 lb/acre of dicamba was applied at planting and 1.5 lb/acre ofdicamba was applied 4 weeks after planting. % inj means percentageinjury on given date. % GR means percentage growth reduction.

Example 6 Controlling Glyphosate Tolerant Weeds by Dicamba

Marestail is one of the major weeds in a crop field. Marestail iseffectively controlled by glyphosate, but the development of methods forcontrolling this common weed with other herbicides is important tominimize opportunities for herbicide tolerance to develop. An analysiswas carried out to determine the extent to which this glyphosatetolerant weed could be controlled by applications of dicamba. Marestail(Conyza canadensis) plants of two biotypes, each from a differentgeographic region, California (CA) and Kentucky (KY), were grown, andtreated at 4-6 inch diameter rosette leaf stage with dicamba asdescribed in Example 2 and 3. The results of the study, as shown inTable 7, demonstrated that dicamba was equally effective in controllingboth susceptible and tolerant biotypes of marestail from CA and KY.Dicamba was more effective in controlling resistant biotypes at lowerapplication rates than glyphosate. For example, 2100 g/ha of glyphosatewas required to obtain about 77% and 91% inhibition of CA and KYresistant biotypes, whereas only 280 g/ha of dicamba was required toobtain about 83% and about 91% control of CA and KY resistant biotypes.

TABLE 7 Control of glyphosate tolerant weeds by dicamba. % Injury (21DAT) Rate MARESTAIL (CA) MARESTAIL (CA) MARESTAIL (KY) MARESTAIL (KY)Formulation g/ha Susceptible Resistant Susceptible Resistant Roundup 84097.2 55.0 76.7 58.3 WeatherMAX ™ 1680 100.0 64.2 97.5 79.2 2100 100.076.7 100.0 90.8 Clarity ™ 50 68.3 61.7 78.3 78.3 140 82.5 80.8 90.0 88.3280 85.0 82.5 91.7 90.8

Example 7 Development of a Method for Controlling Glyphosate TolerantWeeds in a Field

Transgenic seeds having dicamba tolerance are planted in a field thathas been treated with glyphosate before planting the transgenic seeds.The field is then treated with a herbicidally effective amount ofdicamba before or after planting the seeds to control glyphosateresistant weeds. The herbicidally effective amount of dicamba is suchthat the growth of glyphosate resistant weeds is controlled, but is notinjurious to the planted crop as shown in the examples described herein.Thus, transgenic seeds having dicamba tolerance in combination with aneffective amount of dicamba are useful for control of glyphosateresistant weeds. The method may be implemented without delaying plantingof the dicamba tolerant crop plants, thus providing a significantadvance over the prior art, in which dicamba must be appliedsufficiently prior to planting such that the dicamba degrades in theenvironment sufficiently to avoid injury to crop plants.

Example 8 Combination of Dicamba and Glyphosate for ControllingGlyphosate Resistant Weeds to Allow Reduced Herbicide Application Rates

As shown in Table 8, dicamba alone was more effective in controllingresistant biotypes at lower application rates than glyphosate. Further,it has unexpectedly been found that dicamba in combination withglyphosate allows control of glyphosate tolerant and susceptible weedsat lower application rates. For example, whereas 200 g/ha of glyphosatewas able to control only 6% of marestail (KY resistant biotype) at 18DAT and 40 g/ha of dicamba was able to control about 52% of the KYbiotype at 18 DAT, a 200 g/ha glyphosate and 40 g/ha dicamba mixture wasable to control about 79% of the KY biotype at 18 DAT.

In general, any formulation containing dicamba appeared to be moreefficacious than glyphosate alone on the resistant biotype. Also, ingeneral, the following trend in effectiveness of glyphosate to dicambaratio on resistant biotype was found to be true at:4:1>10:1>20:1>40:1>80:1. The results show that a glyphosate to dicambamixture ratio of 4:1 containing 200 g/h glyphosate and 50 g/h dicambaprovided superior control than either glyphosate or dicamba alone.

TABLE 8 Effect of dicamba and glyphosate for controlling glyphosateresistant weeds. % Injury (18 DAT) % Injury (30 DAT) Marestail MarestailMarestail Marestail CHEMICAL Rate Susceptible Resistant SusceptibleResistant FORMULATION g/ha RATIO (KY) (KY) (KY) (KY) RoundupWeatherMAX ™ 200 86.0 5.8 96.3 0.0 400 99.7 25.0 100.0 18.3 800 100.046.7 100.0 44.2 1600 100.0 59.2 100.0 62.5 Clarity ™ 2.5 6.7 10.8 15.87.5 5 18.3 25.0 20.8 35.0 10 34.2 35.8 29.2 39.2 20 40.8 45.8 40.0 45.040 50.0 52.5 51.7 68.3 80 68.3 69.2 71.7 84.3 100 83.3 75.8 86.3 87.5200 89.2 83.3 99.3 94.3 Roundup WeatherMAX ™ +  200 + 2.5 80:1 50.8 20.855.8 31.7 Clarity ™ 400 + 5  80:1 85.8 39.2 97.7 40.8 800 + 10 80:1 99.747.5 100.0 45.0 1600 + 20  80:1 100.0 50.8 100.0 63.3 RoundupWeatherMAX ™ + 200 + 5  40:1 56.7 28.3 64.2 35.0 Clarity ™ 400 + 10 40:182.5 40.0 94.2 43.3 800 + 20 40:1 99.3 53.3 100.0 60.8 1600 + 40  40:1100.0 70.8 100.0 80.8 Roundup WeatherMAX ™ + 200 + 10 20:1 58.3 38.366.7 40.0 Clarity ™ 400 + 20 20:1 81.7 56.7 93.3 50.0 800 + 40 20:1 99.062.5 100.0 73.3 1600 + 80  20:1 99.7 77.5 100.0 88.3 RoundupWeatherMAX ™ + 200 + 20 10:1 56.7 52.5 70.8 60.0 Clarity ™ 400 + 40 10:184.2 79.2 93.3 86.3 800 + 80 10:1 98.7 83.3 100.0 96.8 1600 + 160 10:199.7 89.2 100.0 99.3 Roundup WeatherMAX ™ + 200 + 50  4:1 61.7 79.2 83.587.2 Clarity ™  400 + 100  4:1 89.2 88.3 99.7 98.7  800 + 200  4:1 99.788.3 100.0 99.3 1600 + 400  4:1 100.0 89.7 100.0 100.0

Example 9 Production of Transgenic Seeds Having Dicamba and GlyphosateTolerance

Methods for producing transgenic seeds having glyphosate tolerance areknown in the art and such seeds can be produced by persons of skill inthe art by using a polynucleotide encoding glyphosate resistant5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) as described in U.S.Pat. No. 5,627,061, U.S. Pat. No. 5,633,435, U.S. Pat. No. 6,040,497 andin U.S. Pat. No. 5,094,945, WO04074443 and WO04009761, all of which arehereby incorporated by reference. Soybean breeding lines containing theRoundup Ready® trait event 40-3-2 (Padgette et al., 1995) have beenproduced. Seeds from soybean plant designated as MON19788 have beendeposited under ATCC Accession No. PTA-6708.

Glyphosate tolerant plants can also be produced by incorporatingpolynucleotides encoding glyphosate degrading enzymes such as glyphosateoxidoreductase (GOX, U.S. Pat. No. 5,463,175, herein incorporated byreference), a glyphosate-N-acetyl transferase (GAT, U.S. Patentpublication 20030083480, herein incorporated by reference), and aglyphosate decarboxylase (WO05003362; US Patent Application 20040177399,herein incorporated by reference).

Dicamba tolerant plants are disclosed herein. A suitable line from eachis crossed and progeny seeds screened with herbicide applications ofglyphosate and dicamba to obtain progeny expressing both genes andexhibiting tolerance to both dicamba and glyphosate. Alternatively,coding sequences conferring tolerance to one or both of the herbicidesare directly introduced into a given line. Seeds from these plants areused for developing a method for controlling weed resistance developmentin a field as described below.

Transgenic seeds having dicamba and glyphosate tolerances were testedfor their tolerance to dicamba, glyphosate, or both herbicides. Table 9shows tolerance of transgenic soybeans carrying glyphosate and dicambatolerance transgenes to glyphosate, dicamba, and glyphosate and dicambaat various stages of plant growth. Injury was not seen on plants wheneither or both herbicides were applied at pre-emergence stage.Post-emergence treatments of either or both herbicides at V3, R1, andR3-4 showed only little injury.

TABLE 9 Tolerance of transgenic soybeans carrying glyphosate and dicambatolerance transgenes to glyphosate, dicamba, and glyphosate and dicamba.Post-emergence Pre-emergence treatment treatment V3 R1 R3-4 Rate 20 DAT8 DAT 7 DAT 18 DAT Plant Line Herbicide Applied gm ae/ha % injury(Average of 4 replications) Non-transgenic Control CLARITY 561 99.0 83.871.3 85.0 RWMax 841 0.0 81.3 66.3 67.5 CLARITY + RWMax 561 + 841 99.593.8 81.3 99.0 RR1 + DMO Line1 CLARITY 561 0.0 7.0 6.3 4.5 RWMax 841 0.03.5 3.5 11.3 CLARITY + RWMax 561 + 841 0.0 3.0 4.0 10.0 RR1 + DMO Line 2CLARITY 561 0.0 5.3 6.3 5.3 RWMax 841 0.0 4.5 4.5 11.7 CLARITY + RWMax561 + 841 0.0 5.0 4.0 8.8 RR1 + DMO Line 3 CLARITY 561 0.0 9.0 8.8 7.5RWMax 841 0.0 3.5 4.0 11.3 CLARITY + RWMax 561 + 841 0.0 4.5 3.5 10.0RR1 + DMO Line 4 CLARITY 561 0.0 8.5 8.8 3.5 RWMax 841 0.0 3.5 3.5 11.3CLARITY + RWMax 561 + 841 0.0 4.5 4.5 8.8 RR2 + DMO Line1 CLARITY 5610.0 8.5 6.3 5.3 RWMax 841 0.0 3.5 3.5 3.0 CLARITY + RWMax 561 + 841 0.05.0 4.5 5.0 RR2 + DMO Line 2 CLARITY 561 0.0 9.0 6.3 3.0 RWMax 841 0.03.5 6.3 3.0 CLARITY + RWMax 561 + 841 0.0 9.5 7.0 3.0 RR2 + DMO Line 3CLARITY 561 0.0 9.5 7.5 3.5 RWMax 841 0.0 3.5 6.3 4.5 CLARITY + RWMax561 + 841 0.0 8.5 3.5 3.3 RR2 + DMO Line 4 CLARITY 561 0.0 5.3 5.8 3.0RWMax 841 0.0 16.5 17.0 4.0 CLARITY + RWMax 561 + 841 0.0 11.0 3.5 5.3

Example 10 Development of a Method for Controlling Weed ResistanceDevelopment in a Field

Transgenic seeds having dicamba and glyphosate tolerance prepared asdescribed above are planted in a field. The field is treated withdicamba and glyphosate before or after planting the seeds using amixture of dicamba and glyphosate in an effective amount to control weedgrowth. Typically about a 1× application rate of either herbicide willbe effective in controlling weed growth, but the rate may be varieddepending upon environmental conditions and the type of weeds beingcontrolled, as is known in the art. The rate of application may also beincreased or decreased depending upon the rate of control desired.Generally speaking, increasing the rate of one herbicide will allow adecrease in the rate of the second herbicide in order to obtain the samelevel of seed control. In specific embodiments, an application of fromabout 200 to about 1600 g/ha of glyphosate is combined with from about20 to about 400 g/ha of dicamba.

A desired application rate may be optimized in any particularenvironment or in the context of a particular weed can be determinedusing the experimental layout of Example 9 with the differentformulation rates described therein. In addition to desired level ofweed control, the herbicide level is selected to avoid using moreherbicide than is needed on the one hand, and to avoid poor weed controlthat could lead to herbicide tolerant plants. Over application ofherbicides could also damage herbicide tolerant crop. As shown inExample 9 above however, combining optimized applications of theseherbicides provides significant levels of control of even herbicidetolerant weeds, and thus represents a major advance in the art.

Example 11 Development of a Method for Controlling Weeds in a SinglePass in a Field

The procedures in Examples 9 and 10 are applied to develop a method forcontrolling weed growth in a crop-growing environment involving plantinga transgenic seed in a field containing a weed or a seed thereof andtreating the field in a single pass though the field. The treatmentcomprises a herbicidally effective amount of dicamba, glyphosate, or amixture thereof, administered contemporaneously with the planting of theseed. The planting, treating, and growing of the transgenic seed areachieved by standard agricultural methods.

Such a method of planting the transgenic seed and treating thetransgenic seed in one pass eliminates the need for a farmer to makemultiple passes through the field, including once for planting and oncefor spraying. The technique therefore reduces fuel and wear-tear coststo farmers.

Example 12 Tolerance of Plants Containing DMO-Encoding PolynucleotideMolecule to Other Auxin-Like Herbicides

Herbicide drift and contamination of herbicide delivery equipment is aserious concern in agriculture and can injure non-target crops resultingin losses to farmers. However, some level of drift is often inevitabledue to changing environmental conditions such as wind and the proximityof growing fields. Further, it is often difficult and expensive toeliminate all residual levels of a herbicide in a tank followingherbicide application and residual herbicides often result ininadvertent injury to crops. Often several rinses of herbicide deliveryequipment are required before it can be used for another herbicide,which wastes water and cleaning chemicals.

As herbicides such as 2,4-D and MCPA are post-emergent herbicides forsome crops, but can cause serious damage to non-target crops, residualcontamination with these herbicides is of particular concern. Atransgenic crop tolerant to at least low levels of these herbicideswould therefore be of significant value in managing injuries due tospray drift and contamination of herbicide equipment. This could alsoreduce the extent of equipment washing needed for herbicide deliveryequipment.

An analysis was therefore carried out to determine whether soybeanplants having DMO-encoding polynucleotide could deactivate otherauxin-like herbicides in addition to dicamba, including 2,4-D and MCPA.This was carried out by applying various concentrations of commerciallyavailable formulations of other auxin-like herbicides such as 2,4-D(Helena, Collierville, Tenn.), MCPA (Agriliance, St. Paul, Minn.),triclopyr (GARLON 3A; Dow Elanco, Indianapolis, Ind.), clopyralid(STINGER; Dow Elanco, Indianapolis, Ind.), picloram (TORDON 22K; DowElanco, Indianapolis, Ind.), or Banvel or CLARITY (BASF, Raleigh, N.C.)to DMO containing plant tissues or plants.

Transgenic soybean plants were obtained by Agrobacterium-mediatedtransformation of soybean explants with a DMO-encoding polynucleotide asdescribed above for the events designated Events 1-4. A non-transgenicline was used as a control. Non-transgenic and transgenic soybean seedswere planted into 3.5-inch square plastic pots containing Redi-earth™(Scotts-Sierra Horticultural Products Co., Marysville, Ohio). The potswere placed on capillary matting in 35 inch×60 inch fiberglass wateringtrays for overhead and/or sub-irrigation for the duration of the testperiod so as to maintain optimum soil moisture for plant growth. Thepots were fertilized with Osmocote (14-14-14 slow release; Scotts-SienaHorticultural Products Co., Marysville, Ohio) at the rate of 100gm/cu.ft. to sustain plant growth for the duration of greenhouse trials,and grown in greenhouses at 27°/21° C. day/night temperature, withrelative humidity between 25%-75% to simulate warm season growingconditions of late spring. A 14 h minimum photoperiod was provided withsupplemental light at about 600 μE as needed.

All herbicide applications were made with the track sprayer using aTeejet 9501E flat fan nozzle (Spraying Systems Co, Wheaton, Ill.) withair pressure set at a minimum of 24 psi (165 kpa). The spray nozzle waskept at a height of about 16 inches above the top of plant material forspraying. The spray volume was 10 gallons per acre or 93 liters perhectare. Applications were made when plants had reached V-3 stage. Alltrials were established in a randomized block design (randomized byrate) with 4 to 6 replications of each treatment depending on plantquality, availability and to account for any environmental variabilitythat may have occurred within the confines of each greenhouse.

All treated plants in greenhouse trials were visually assessed at about4, 14, 18, and 21 days after treatment (DAT) for injury on a scale of 0to 100 percent relative to untreated control plants, with zerorepresenting “no” injury and 100% representing “complete” injury ordeath. Data were collected using a palm top computer and analyzed usingstandard statistical methods. The results shown in Table 10 clearlyindicate tolerance of transgenic soybean to other auxin-like herbicidessuch as 2,4-D and MCPA relative to the non-transgenic line.

TABLE 10 Percentage injury relative to un-treated controls at 25 DATpost-V3 applications of different auxin-like herbicides tonon-transgenic or transgenic soybean plants.* Herbicide Plant/trial 280561 1120 % injury at shown rates (g ae/ha**) at 21 DAT DicambaNon-transgenic 100 100 (Clarity) Event 1 0.0 1.2 Event 2 0.0 1.7 Event 30.0 0.7 Event 4 0.0 1.5 Dicamba Non-transgenic 100.0 100.0 (Banvel)Event 1 0.0 1.5 Event 2 0.0 0.7 Event 3 0.0 0.5 Event 4 0.0 1.3 2,4-DNon-transgenic 86.8 100.0 100.0 Event 1 58.3 75.0 100.0 Event 2 64.294.7 100.0 Event 3 40.0 85.0 100.0 Event 4 45.8 84.2 100.0 MCPANon-transgenic 93.0 98.3 100.0 Event 1 72.5 99.3 100.0 Event 2 55.0 95.099.7 Event 3 55.0 95.8 100.0 Event 4 88.3 98.8 100.0 LSD 16.3 10.6 3.7 %injury shown rates (g ae/ha**) at 14 DAT Triclopyr Non-transgenic 86.797.3 98.7 Event 1 88.3 95.7 99.3 Event 2 86.7 98.7 99.3 Event 3 86.794.0 96.3 Event 4 90.8 98.0 99.2 Clopyralid Non-transgenic 99.3 100.0100.0 Event 1 99.2 100.0 100.0 Event 2 98.2 99.7 100.0 Event 3 99.3100.0 100.0 Event 4 99.7 100.0 100.0 Picloram Non-transgenic 99.3 100.0100.0 Event 1 99.7 100.0 100.0 Event 2 99.3 100.0 100.0 Event 3 99.399.7 100.0 Event 4 99.3 100.0 100.0 % injury at shown rates (g ae/ha**)at 21 DAT LSD 2.9 1.8 1.4 *The % injury was represented as ANOVA meancomparisons. **grams of active acid equivalent/hectare

Another auxin-like herbicide Butyrac 200 (2,4-DB; Albaugh) was alsotested on transgenic soybean plants carrying a DMO gene for testing theplants tolerance to it. The herbicide was applied as a post-emergencetreatment at three application rates on two transgenic soybean eventsand compared with a non-transgenic line for total crop injury across allthree application rates: 280 g/ha (0.25 lb/a), 561 g/ha (0.5 lb/a) and841 g/ha (0.75 lb/a) (see Table 11). Both transgenic soybean linesshowed low level of tolerance to 2,4-DB. This example shows that dicambatolerant soybean is also tolerant to low levels of 2,4-DB and should beuseful in managing damage from spray drift from the same or neighboringfields to prevent crop loses, and would exhibit tolerance to residuallevels of 2,4-DB following incomplete washing of herbicide deliveryequipment.

TABLE 11 Percentage injury relative to the untreated control at 16 DATby the application of 2,4-DB to non- transgenic or transgenic soybeanplants. % injury at shown rates (g ae/ha) at 16 DAT Herbicide Plant 280561 1120 2,4-DB Non- 59.2 70.0 79.2 (Butyrac 200) transgenic NE3001462-1-21 25.0 43.3 75.8 469-13-19 18.3 37.5 70.0

This example shows that transgenic soybean plants exhibit tolerance toother auxin-like herbicides, indicating a likely common deactivationmechanism for dicamba and other auxin-like herbicides such as 2,4-D andMCPA. In case of triclopyr, clopyralid, and picloram, the applicationrate of 280 g ae/ha appeared too stringent in this study and thus lowerconcentrations may be desired in most settings to reduce plant damage.Thus, a DMO polynucleotide containing soybean that is tolerant todicamba is also tolerant to low levels of 2,4-D and MCPA and shouldprevent or minimize damage from spray drift from same or neighboringfields to prevent crop loses, and would exhibit tolerance to residuallevels of these herbicides following incomplete washing of herbicidedelivery equipment. The herbicide delivery equipment could include atank, container, hose, strainer, boom, sprayer, nozzle, pump, andaccessories such as coupling, elbows, shanks, and valves. The deliveryequipment is operable manually or mechanically for example on a farmvehicle, airplane, and helicopter, among others.

Example 13 Production of Dicamba Tolerant Transgenic Corn Plants

To test the use of a DMO gene in providing dicamba tolerance tomonocots, transgenic corn plants were produced that comprise a DMO geneas disclosed above with or without a transit peptide (e.g. TaWaxy, CTP1,CTP2synthetic, CTP4) under the control of plant gene expression elementssuch as a promoter (e.g. PC1SV, e35S, OsAct1, OsTPI, OsAct15), and anintron (e.g. OsAct1, OsAct15, OsTPI, ZmHSP70). This expression elementcontains first intron and flanking UTR exon sequences from the riceactin 1 gene and includes 12 nt of exon 1 at the 5′ end and 7 nt of exon2 at the 3′ end), and a 3′UTR (e.g. TaHsp17). Nucleotide sequences/an orpatent references for various expression elements are disclosed inco-pending application U.S. Ser. No. 60/891,675.

Transgenic corn plants were produced by the methods known in the artsuch as WO9506722 and US patent application 20040244075. Transgenic cornevents having single copy were evaluated for dicamba tolerance at asingle location replicated trial. Six events from each of the sixconstructs were used. The experimental design was as follows:rows/entry: 1; treatment: 0.5 lb/a of dicamba at V3 stage followed by 1lb/a of dicamba at V8 stage (Clarity®, BASF, Raleigh, N.C.);replications: 2; row spacing: 30 inches; plot length: minimum 20 feet;plant density: about 30 plants/17.5 ft.; alleys: 2.5 feet. The entireplot was fertilized uniformly to obtain an agronomically acceptablecrop. A soil insecticide such as Force® 3G (Syngenta Crop Protection,Greensboro, N.C., USA) at 5 oz. per 1000 ft. of row for control of cornrootworm was applied at planting time. If black cutworm infestation wasobserved, POUNCE® 3.2EC at 4 to 8 oz. per acre rate (FMC Corporation,Philadelphia, Pa.) was used. In addition, an insecticide spray programwas used to control all above ground lepidopteran pests includingEuropean corn borer, corn earworm, and fall armyworm. POUNCE® 3.2EC at 4to 8 oz. per acre was applied every 3 weeks to control lepidopteranpests; about 4 applications were made. The plot was kept weed free witha pre-emergence application of a herbicide such as Harness® Xtra 5.6 L(Monsanto, St. Louis, Mo.) and Degree Xtra® (Monsanto, St. Louis, Mo.).If weed escapes were observed in the untreated check, they werecontrolled by hand weeding or a post-emergence application of PERMIT(Monsanto, St. Louis, Mo.) or BUCTRIL® (Bayer, Research Triangle Park,N.C.) over the entire trial.

Corn inbred lines transformed with DNA constructs comprising a DMOtransgene were tested for dicamba tolerance by measuring brace rootinjury when treated with 0.5 lb/a of dicamba at V3 stage followed by 1lb/a of dicamba at V8 stage. Brace root injury was evaluated visually bycounting the number of plants in a row showing an “atypical” morphologyof having the brace roots fused as compared to a typical morphology of“finger-like” structure. As shown in Table 12, corn plants transformedwith DNA constructs coding for a DMO without linking it to a CTP(pMON73699, pMON73704) showed higher level of brace root injury, i.e.lower level of protection upon dicamba treatment. The constructs codingfor a DMO linked to a CTP (pMON73716, pMON73700, pMON73715, pMON73703)showed lower level of brace root injury, i.e. higher level of protectionupon dicamba treatment.

TABLE 12 Percentage brace root injury as a measure of dicamba toleranceexhibited by transgenic corn plants transformed with DNA constructscarrying DMO. Brace Inbreds/ root Constructs Details injury 01CSI6Susceptible inbred to dicamba 95.4 LH244 Resistant inbred to dicamba93.8 pMON73699 PC1SV/I-OsAct1/DMO-Wmc/TaHsp17 93.2 pMON73704e35S/I-OsAct1/DMO-Wmc/TaHsp17 91.3 pMON73716PC1SV/I-OsAct1/TaWaxy/DMO-Wmc/TaHsp17 78.8 pMON73700PC1SV/I-OsAct1/CTP1/DMO-Wmc/TaHsp17 74.4 pMON73715PC1SV/I-OsAct1/CTP2syn/DMO-Wmc/TaHsp17 68.2 pMON73703e35S/I-OsAct1/CTP1/DMO-Wmc/TaHsp17 68.8

Example 14 Production of Dicamba Tolerant Transgenic Cotton Plants

To test the use of DMO gene in providing dicamba tolerance to cotton,transgenic cotton plants were produced. Several DNA constructs carryinga DMO coding region as disclosed herein with a transit peptide (e.g.,PsRbcS CTP, CTP1, CTP2) under the control of plant gene expressionelements such as a promoter (e.g. PC1SV, FMV, or e35S), and a 3′UTR(e.g. E6; Accession #U30508) were produced and transformed into cotton(Gossypium hirsutum) as follows. Nucleotide sequences/and or patentreferences for various expression elements are disclosed in co-pendingapplication U.S. Ser. No. 60/891,675. Media used are noted in Table 13.

Cotton transformation was performed, for instance as described accordingto U.S. Patent Application Publication 20040087030, via an embryogenicapproach. Explants of cotton cv Coker 130 were grown in vitro and with aliquid suspension of Agrobacterium tumefaciens carrying a DNA constructof interest, using selection on kanamycin containing media. Putativetransgenic plantlets were then transferred to soil to obtain maturecotton plants. The transgenic nature of transformants was confirmed byDNA testing.

TABLE 13 Composition of various media used for cotton transformation.Amount/L Components Glucose Sucrose UMO TRP+ SHSU MS basal salts(Phytotech.) 4.33 g 4.33 g 4.33 g 4.33 g — Gamborg's B5 vitamins 2 ml 2ml 2 ml 2 ml — (Phytotech) (500X) 2,4-D (1 mg/ ml) 0.1 ml 0.1 ml — —Stewart and Hsu majors (10X) — — — — 100 ml Stewart and Hsu minors(100X) — — — — 10 ml Steward and Hsu organic (100X) — — — — 10 mlKinetin (0.5 mg/ ml) 1 ml 1 ml — — — Chelated iron (100X) — — — — 1.5 mlGlucose 30 g 30 g 30 g 30 g 5 g Potassium nitrate — — — 1.9 g — Caseinhydrolysate — — — 0.1 g — pH 5.8 5.8 5.8 5.8 6.8 Phytagel (Sigma) 2.5 g2.5 g — — — Gelrite (Kelco) — — 3.5 g 3.5 g 2.2 g Carbenicillin (250 mg/ml) 1.7 ml 1.7 ml 1.7 ml 1.7 ml — Cefotaxime (100 mg/ ml) 1 ml 1 ml 1 ml1 ml — Benlate (50 mg/ ml) — — — 1 ml 1 ml Kanamycin (50 mg/ ml) 0.8-1.0ml 0.8-1.0 ml 1 ml — — Sucrose — 0.1 g — — — Ascorbic acid — — 100 mg

Transformed cotton plants that comprise a DNA construct, i.e, eachcomprising a different combination of a DMO coding region with a transitpeptide, a promoter, and a 3′UTR, were treated with dicamba (Clarity®,BASF, Raleigh, N.C.) as a post-emergent treatment at V4-5 growth stageat the rate of 561 g ae/ha (0.5 lb/a) and found to be tolerant whereasuntransformed cotton plants showed an injury rate of 79% to 86%.Transgenic plants showing more than 95% tolerance (equal to less than 5%injury) were selected for further studies. Transgenic plants were alsotolerant to a subsequent post-emergent treatment of dicamba. Forexample, the plants that were treated with 0.5 lb/acre of dicamba atV3-4 stage followed by either 1 or 2 lb/acre of dicamba at V5 or laterstages were still tolerant to dicamba. R1 transgenic seeds and plantswere also subjected to pre-emergence or pre-emergence and post-emergencedicamba treatment and found to be tolerant. This example shows that aDMO gene can provide dicamba tolerance to cotton at various stages ofgrowth thus enabling application of dicamba at various stages to obtaineffective weed control.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. More specifically, it will beapparent that certain agents that are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

REFERENCES

The references listed below are incorporated herein by reference to theextent that they supplement, explain, provide a background for, or teachmethodology, techniques, and/or compositions employed herein.

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1-17. (canceled)
 18. A method for controlling weed growth in acrop-growing environment comprising: a) applying a herbicidallyeffective amount of an auxin-like herbicide to a crop-growingenvironment; b) planting a transgenic seed of a monocotyledonous plantexpressing a nucleic acid encoding dicamba monooxygenase in soil of thecrop-growing environment within 15 days of applying the auxin-likeherbicide, wherein the herbicidally effective amount is an amount thatdoes not damage the transgenic seed or a plant that germinates therefrombut will damage a seed or a plant that germinates therefrom of the samegenotype that lacks the nucleic acid and is planted under the sameconditions as the transgenic seed; and c) allowing the seed to germinateinto a plant.
 19. The method of claim 18, wherein the nucleic acid isselected from the group consisting of (1) a nucleic acid sequenceencoding the polypeptide of SEQ ID NO:8, (2) a nucleic acid sequencecomprising the sequence of SEQ ID NO:7, (3) a nucleic acid sequence thathybridizes to a complement of the nucleic acid sequence of SEQ ID NO:7under conditions of 5×SSC, 50% formamide and 42° C., (4) a nucleic acidsequence having at least 70% sequence identity to the nucleic acidsequence of SEQ ID NO:7, and (5) a nucleic acid sequence encoding apolypeptide having at least 70% sequence identity to the polypeptidesequence of SEQ ID NO:8
 20. The method of claim 18, wherein theherbicide is applied prior to, concurrently with, or after the plantingof the seed.
 21. The method of claim 18, wherein the transgenic seed isplanted in the soil within about 12, 10, 7, or 3, days before or afterthe herbicide is applied.
 22. The method of claim 18, wherein thetransgenic seed germinates from between about 18 days and 0 days aftertreating the soil.
 23. The method of claim 18, wherein the transgenicseed germinates from between about 14 days and 0 day after treating thesoil.
 24. The method of claim 18, wherein the transgenic seed germinatesfrom between about 7 days and 0 days after treating the soil.
 25. Themethod of claim 18, wherein the auxin-like herbicide is selected fromthe group consisting of a phenoxy carboxylic acid compound, benzoic acidcompound, pyridine carboxylic acid compound, quinoline carboxylic acidcompound, and benazolinethyl compound.
 26. The method of claim 25,wherein the phenoxy carboxylic acid compound is2,4-dichlorophenoxyacetic acid, (4-chloro-2-methylphenoxy)acetic acid(MCPA), or 4-(2,4-dichlorophenoxy)butyric acid (2,4-DB).
 27. The methodof claim 26, wherein the herbicidally effective amount of2,4-dichlorophenoxyacetic compound is from about 280 g/ha to about 1200g/ha, or the herbicidally effective amount of4-(2,4-dichlorophenoxy)butyric acid (2,4-DB) is less than 1120 g/ha. 28.The method of claim 26, wherein the herbicidally effective amount of2,4-dichlorophenoxyacetic compound is at least about 590 g/ha.
 29. Themethod of claim 25, wherein the phenoxy carboxylic acid compound is(4-chloro-2-methylphenoxy)acetic acid (MCPA).
 30. The method of claim29, wherein the herbicidally effective amount of MCPA is at least about200 grams/hectare.
 31. The method of claim 29, wherein the herbicidallyeffective amount of MCPA is from about 500 g/ha to about 1120 g/ha. 32.The method of claim 25, wherein the benzoic acid compound is dicamba.33. The method of claim 32, wherein the herbicidally effective amount ofdicamba is at least about 175 g/ha.
 34. The method of claim 32, hereinthe herbicidally effective amount of dicamba is from about 250 g/ha toabout 600 g/ha.
 35. The method of claim 18, wherein the monocotyledonousplant is selected from the group consisting of corn, rice, sorghum,wheat, rye, millet, sugarcane, oat, triticale, switchgrass, andturfgrass.
 36. The method of claim 35, wherein the monocotyledonousplant is a corn or sorghum plant.
 37. The method of claim 18, furthercomprising applying a second treatment of an auxin-like herbicide afterthe seed germinates.
 38. The method of claim 37, wherein the secondtreatment is carried out at a time selected from the group consisting ofbetween about the V1 to V2 and V3 to V4 stages, before flowering, atflowering, after flowering, and at seed formation.
 39. A method forcontrolling a glyphosate tolerant weed in a field comprising: a)planting a transgenic seed in a field comprising a glyphosate tolerantweed or a seed thereof, wherein the seed comprises a transgeneconferring glyphosate tolerance and a transgene encoding dicambamonooxygenase, the transgene encoding dicamba monooxygenase comprising anucleic acid sequence selected from the group consisting of (1) anucleic acid sequence encoding the polypeptide of SEQ ID NO:8, (2) anucleic acid sequence comprising the sequence of SEQ ID NO:7, (3) anucleic acid sequence that hybridizes to a complement of the nucleicacid sequence of SEQ ID NO:7 under conditions of 5×SSC, 50% formamideand 42° C., (4) a nucleic acid sequence having at least 70% sequenceidentity to the nucleic acid sequence of SEQ ID NO:7, and (5) a nucleicacid sequence encoding a polypeptide having at least 70% sequenceidentity to the polypeptide sequence of SEQ ID NO:8; b) growing the seedinto a plant; and c) treating the field with an amount of an auxin-likeherbicide and glyphosate effective to control weed growth of theglyphosate tolerant weed, wherein the seed is from a monocotyledonousplant. 40-43. (canceled)
 44. The method of claim 39, wherein themonocotyledonous plant is selected from the group consisting of corn,rice, sorghum, wheat, rye, millet, sugarcane, oat, triticale,switchgrass, and turfgrass.
 45. The method of claim 44, wherein themonocotyledonous plant is a corn or sorghum plant. 46-75. (canceled) 76.A method for controlling weed growth in a crop-growing environmentcomprising: a) planting a transgenic seed in a field comprising a weedor a seed thereof, wherein the transgenic seed comprises a transgeneconferring glyphosate tolerance and a transgene conferring dicambatolerance; b) treating the field with a herbicidally effective amount ofdicamba, glyphosate, or a mixture thereof, wherein the planting and thetreating is done in a single pass through the field; and c) growing thetransgenic seed into a plant wherein the transgenic seed is from amonocotyledonous plant. 77-81. (canceled)
 82. The method of claim 76,wherein the monocotyledonous plant is selected from the group consistingof corn, rice, sorghum, wheat, rye, millet, sugarcane, oat, triticale,switchgrass, and turfgrass seed.
 83. The method of claim 82, wherein themonocotyledonous plant is a corn or sorghum plant. 84-85. (canceled)